This play is full of so many possible themes - you could have a complete field day in answering this question as an essay. But one overarching question seems to link all of these possible themes together - what is the nature of our existence as human beings?
We enter the world with no sense of identity and gradually as we grow up assume our identity from things around us - our families, our achievements etc. However, one of the major aspects of the play seems to point out that our assumed identity maybe based on illusory concepts. Beckett himself rejected the church as an "illusion" and believed that man's greatest achievements, when considered in the context of the whole universe, count for nothing, but at the same time, life without illusions of grandeur and importance would be a very sad affair.
In relation to this, you need to ask yourself what kind of world it is that Vladimir and Estragon live in. They don't have the normal securities that we do in our lives - nothing is certain, violence can appear be inflicted upon them at any time, and there are no concepts such as justice or securities of any afterlife. Even simple tasks are made out to be major achievements. The human condition is therefore depicted to be incredibly insecure, but perhaps the one redeeming feature is our search for meaning and significance.
Against this relentless nihilism of the play, another redeeming aspect is the friendship of Vladimir and Estragon, and it is worth the pain to examine their relationship based in the context of the whole play. What does their friendship say about hope in an otherwise hopeless environment? What hope does it offer to us as human beings as we struggle to make sense of who we are?