The North and South started to be different from the very start of colonization. The South (outside of the backwoods areas) had an economy that was based on unfree labor. It soon developed an economic system of large landowners with unfree (at first indentured servants, then slaves) workers. Meanwhile, the North's economy was coming to be based on small farms and some cities with merchants and artisans.
This led to the other differences between North and South. It led, for example, to the North being more egalitarian and the South being more aristocratic. It led to the North wanting tariffs and the South hating them. This basic difference in the basis of the regional economies made the two regions "take on their different natures."