Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcγ receptor IIB (FcγRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)–FcγRIIB coligation. The function of Ship in lymphocytes was investigated in Ship^−/− recombination-activating gene (Rag)^−/− chimeric mice generated from gene-targeted Ship^−/− embryonic stem cells. Ship^−/−Rag^−/− chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship^−/− splenic B cells displayed prolonged Ca²⁺ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR–FcγRIIB coligation. These results demonstrate that Ship plays an essential role in FcγRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca²⁺ flux and MAPK activation.