Thats a great question. My thesis defense had two "categories" of questions. Those from my thesis committee and those from the audience. The questions from the audience were mainly focused on my background or intro slides and conclusions from my work. Be prepared for random questions from the audience.
My thesis committe questions were focused on the specific assays (experimental questions, how I could have designed a specific experiment differently to get a better result, what other experiments could I have done) and what direction I would move the project if I were to continue to work on it. This last question is particularly important. Think about what the next publication would be. What is your next hypothesis based on the work you completed as a graduate student.