Biggest is probably the product-market fit. Is your product or service helping (enough) people solve a problem or fulfill a need?

This is the biggest question a startup is trying to answer. You have to answer this question within a "finite time" and with "limited resources".

So it really comes down to how focused a startup is in answering this question. If you lose focus with other stuff (co-founder issues, business cards, spending too much time making the product beautiful or whatever else) you are going to run out of time and resources and you have lost the game.

I interview some of the very successful digital entrepreneurs on the web that respond to questions like this. Check out the interviews here: http://treptalks.com