Biochemical studies have indicated that p31^comet and TRIP13 are critical for inactivating MAD2. To address unequivocally whether p31^comet and TRIP13 are required for mitotic exit at the cellular level, their genes were ablated either individually or together in human cells. Neither p31^comet nor TRIP13 were absolutely required for unperturbed mitosis. MAD2 inactivation was only partially impaired in p31^comet-deficient cells. In contrast, TRIP13-deficient cells contained MAD2 exclusively in the C-MAD2 conformation. Our results indicate that although p31^comet enhanced TRIP13-mediated MAD2 conversion, it was not absolutely necessary for the process. Paradoxically, TRIP13-deficient cells were unable to activate the spindle-assembly checkpoint, revealing that cells lacking the ability to inactivate MAD2 were incapable in mounting a checkpoint response. These results establish a paradigm of the roles of p31^comet and TRIP13 in both checkpoint activation and inactivation.