Author Summary The spindle checkpoint protects cells from aneuploidy by monitoring the status of the kinetochore-microtubule attachment. Defects in this checkpoint pathway and in kinetochore-microtubule attachment can cause substantial aneuploidy in cells. The duration of the mitotic arrest induced by the spindle checkpoint is not indefinite: cells eventually exit from mitosis and re-enter interphase. Because continued activation of the spindle checkpoint is lethal, adaptation to the spindle checkpoint arrest is essential so that cells have a chance for survival as opposed to certain death. However, adaptation of the spindle checkpoint has a flip side—adapted cells could have an increased chance of aneuploidy due to premature mitotic exit. Thus, it is essential that this mechanism be regulated appropriately. Despite the importance of understanding the adaptation of the spindle checkpoint, little is known to date about this mechanism. We found that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for adaptation of the spindle checkpoint, a finding providing the molecular insight on how adaptation to prolonged mitotic arrest induced by the spindle checkpoint occurs.