There was only a couple of years between end of the fighting in Kansas and the start of the Civil War. It was still a tense time, as Kansans (and the rest of the nation) were waiting to see if the Wyandotte Constitution would be accepted and Kansas enter the union as free state (which it was in January of 1861).

The Kansas-Nebraska Act threatened the balance in the senate established by the Missouri Compromise. I wouldn't say the Act was the final reason, but rather that it created a situation in Kansas that caused the relations between North and South to deteriorate rapidly. Before the Act, many abolitionists had boycotted elections, refusing to participate in a government that condoned the institution of slavery. When Missourians crossed the border and stole the 1855 territorial election in Kansas, it was a wake-up call to the abolitionists. There was a growing outrage in the North over the election, the drafting of the pro-slavery Lecompton Constitution, and the laws passed by the new pro-slavery territorial government that outlawed abolitionist talk and publication in the territory. This helped propel the new Republican party into the Senate in the 1856 elections. The question of the legitimacy of the Lecompton Constitution also split the Democratic party. Charles Sumner, senator from Massachusetts, denounced the events in Kansas:

here in our republic, force, aye, sir, FORCE — has been openly employed in compelling Kansas to the pollution of slavery, all for the sake of political power.

Two days later, Sumner was attacked in the chamber by the cane-wielding Preston Brooks, congressman from South Carolina. Whatever divides existed before these events, it was clear that supporters of slavery were going to be fanatical in not only their defense of that institution, but in suppressing any talk of ending it.

Charles Robinson, the first governor of Kansas and a leading member of the Free State movement, covers the events in Kansas pretty well in his book The Kansas Conflict.