Pten is a tumor suppressor gene mutated in human cancers. We used the Cre-loxP system to generate a B cell–specific mutation of Pten in mice (bPten^flox/floxmice). bPten^flox/flox mice showed elevated numbers of B1a cells and increased serum autoantibodies. Among B2 cells in bPten^flox/flox spleens, numbers of marginal zone B (MZB) cells were significantly increased while those of follicular B (FOB) cells were correspondingly decreased. Pten-deficient B cells hyperproliferated, were resistant to apoptotic stimuli, and showed enhanced migration. The survival kinase PKB/Akt was highly activated in Pten-deficient splenic B cells. In addition, immunoglobulin class switch recombination was defective and induction of activation-induced cytidine deaminase (AID) was impaired. Thus, Pten plays a role in developmental fate determination of B cells and is an indispensable regulator of B cell homeostasis.