The spindle assembly checkpoint monitors the integrity of the spindle microtubules, which attach to sister chromatids at kinetochores and play a vital role in preserving genome stability by preventing missegregation. A key target of the spindle assembly checkpoint is securin, the separase inhibitor. In budding yeast, loss of securin results in precocious sister chromatid separation when the microtubule spindle is disrupted. However, in contrast to budding yeast, mammalian securin is not required for spindle checkpoint, suggesting that there are redundant mechanisms controlling the dissolution of sister chromatid cohesion in the absence of securin. One candidate mechanism is the inhibitory phosphorylation of separase. We generated a nonphosphorylable point mutant (S1121A) separase allele in securin-/- mouse embryonic stem cells. Securin(-/-)separase(+/S1121A) cells are viable but fail to maintain sister chromatid cohesion in response to the disruption of spindle microtubules, show enhanced sensitivity to nocodazole, and cannot recover from prometaphase arrest.