BCR cross-linking promotes mature B cell proliferation and survival. PI3K-mediated down-regulation of proapoptotic and antimitogenic genes such as forkhead box transcription factor class O 1 (FOXO1) is an important component of this process. Previously, BCR-induced phosphorylation of FOXO1 was shown to lead to a block in nuclear localization and subsequent protein degradation. We demonstrate that the BCR also signals through PI3K to down-regulate FOXO1 mRNA expression. Bruton's tyrosine kinase (Btk), a downstream effector of PI3K, signals through B cell linker protein (BLNK) and phospholipase C (PLC)gamma2 to mediate B cell proliferation and survival in response to BCR cross-linking. BCR-induced down-regulation of FOXO1 mRNA was impaired in murine knockouts of Btk, BLNK, and PLCgamma2. Because B cells in these models are predominantly immature, experiments were also performed using mature B cells expressing low levels of Btk and BLNK. Similar results were obtained. Inhibitors of downstream components of the Btk/BLNK/PLCgamma2 pathway were used to define the mechanism by which Btk signaling inhibits FOXO1 expression. The protein kinase Cbeta inhibitor Gö6850 had minimal effects on BCR-mediated FOXO1 mRNA down-regulation. However, cyclosporin A, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, had similar effects on FOXO1 mRNA expression as the PI3K inhibitor LY294002. Neither Btk deficiency nor cyclosporin A prevented FOXO1 protein phosphorylation, indicating that PI3K down-regulates FOXO1 via two independent pathways. We show that the Btk/BLNK/PLCgamma2 pathway mediates BCR-induced changes in expression of the FOXO1 target gene cyclin G2. These observations support the hypothesis that Btk mediates BCR-induced proliferation and survival in part via inhibition of FOXO expression.