Inactivation of maturation-promoting factor [(MPF) Cdk1/Cyclin B] is a key event in the exit from mitosis. Although degradation of Cyclin B is important for MPF inactivation, recent studies indicate that Cdk1 phosphorylation and inactivation occur before Cyclin B degradation and, therefore, also may be important steps in the exit from mitosis. Cdk1 activity is controlled by the Cdc25C phosphatase, which is turned on at the G(2)/M transition to catalyze Cdk1 activation. PP2A:B56delta is a negative regulator of Cdc25C during interphase. We show here that PP2A:B56delta also regulates Cdc25C at mitosis. Failure of PP2A:B56delta to dephosphorylate Cdc25C at mitosis results in prolonged hyperphosphorylation and activation of Cdc25C, causing persistent dephosphorylation and, hence, activation of Cdk1. This constitutive activation of Cdc25C and Cdk1 leads to a delayed exit from mitosis. Consistent with Cdk1 as a major biological target of B56delta, stable knockdown and germ-line mouse KO of B56delta leads to compensatory transcriptional up-regulation of Wee1 kinase to oppose the Cdc25C activity and permit cell survival. These observations place PP2A:B56delta as a key upstream regulator of Cdk1 activity upon exit from mitosis.