One important theme in Of Mice and Men is loneliness. Many characters in the novel reveal this theme. George states early on in the novel that the life of a migrant farmer or ranch hand is a lonely life. That's one of the reasons he keeps Lennie by his side: to aid in his loneliness. In the end his loneliness is imminent as his hope for companionship dies with Lennie. Crooks, Curley's wife, and Candy are make reference to the loneliness they feel throughout the novel as well.
Another important theme in Of Mice and Men is the importance of dreams...or even the quest for the "American Dream." Many of the characters have a dream for their lives other than what they are actually living. These dreams, however, are ultimately unattainable. George and Lennie and eventually Candy have the dream of owning their own farm--a dream that gives them life and keeps them going, but ultimately is impossible. Curley's wife confesses that she has a dream of being a movie star, but literally only dreams of this and has resigned herself to life on the farm.
It is difficult to be certain of the two most important themes in John Steinbeck's Of Mice and Men because there are several that are extremely meaningful. This question is subjective, so this is what I think.
The first theme I find central to the story is Idealism vs Reality. The second that strikes a chord with me is Friendship. (Others that are central to the story as well are: loyalty, mental disability, class conflict, alienation and loneliness, and race and racism.)
"Idealism and Reality" can be seen in the plans that Lenny and George make about having their own place. This, during the Great Depression, when so many people were disenfranchised, becomes their American Dream: to have a place of one's own without the need for be forever moving from one place to another without the ability to put down roots.
For Lenny, the idea of having a place to raise rabbits is his one goal in life: for George has promised that he can care for the rabbits. For George, it is a dream of staying in one place and not having to be fearful of trouble Lennie might get into which would force them to move on. It also allows him to dream of a life where he works for himself rather than someone else.
Having such a spread is idealism on the men's part. Crooks, the black stable hand, is quick to point out the foolishness of their dream:
Nobody never gets to heaven, and nobody gets no land. It's just in their head. They're all the time talkie' about it, but it's jus' in their head.
However, the reality is that being able to afford such a home requires money which comes from work. Work during the Depression is hard to find, and it seems that the dream may never come true, especially after Lennie accidentally kills Curley's wife.
The second theme is "friendship." George promised Lennie's aunt, as she was dying, that he would take care of Lennie who has a mental disability. A tall man, Lennie is unaware of his own strength, and sometimes acts inappropriately. George is forever correcting him, yelling at him, warning him, and watching out for Lennie. Lennie depends a great deal on George and tries his best to listen to what George tells him to do, when he can remember. George is Lennie's only friend, and George gets impatient with Lennie, but also seems to sincerely care about him. It is difficult in many ways to separate "friendship" from "loyalty," as both men are extremely loyal to each other. When Lennie kills Curley's wife, it is George who knows he must kill Lennie. Lennie has the heart and soul of a child, and George wants to spare him the horror of being caught, beaten, and most likely lynched or shot. This is very difficult for George, but his friendship with Lennie requires him to do what he can for the man he has traveled with for so long. So he tells Lennie to imagine the home they will one day have together: it is then that he kills Lennie. George provides the most compassionate death he can rather than have Lennie face the brutality of the men pursuing Lennie who could never understand his mental deficiency.
Hope is something the two men share, and allows them...
to...dream, but the reality of their brutal life destroys the dream and the friendship.
Given that the question of themes in any work is subjective, here is what I believe to be the two most important themes in Of Mice and Men:
Loneliness: Many of the characters in the story are lonely. Lennie could be considered to be lonely given his inability to truly connect with the other characters because of his diminished mental capacity. Candy is a lonely character because he cannot work in the same capacity that the other ranchers can, due to the loss of his hand. Crooks is lonely given that he is African American and is, therefore, isolated from the other characters. Curley's wife, the only one to actually name her loneliness, is isolated given the fact that the ranchers see her as problematic-she causes trouble for the ranchers between them and her husband. Many of the characters, although all part of community, feel loneliness based upon their inability to create true relationships.
Dreams: Perhaps the most important theme of the novel is that of having a dream. During the time of the novel, many people were facing financial hardships. The novel explicitly illustrates this theme through the continuous repetition of Lennie and George's "dream". Like many people at the time, and even today, the chance to change one's future for the better is the only thing that allows one to progress through life. For Lennie and George, this dream functions no differently. It is the dream which keeps Lennie and George focusing on the future- the dream of owing their own farm and being their own bosses. This theme keeps the novel relevant given that today many people can still relate to this.