Spi‐B is a hematopoietic‐specific Ets family transcription factor closely related to PU. 1. Previous gene targeting experiments have shown that PU. 1 is essential for the production of both lymphocytes and monocytes. We have now generated mice with a null mutation at the Spi‐B locus. Unlike PU. 1 mutant mice, Spi‐B−/− mice are viable, fertile and possess mature B and T lymphocytes. However, Spi‐B−/− mice exhibit severe abnormalities in B cell function and selective T cell‐dependent humoral immune responses. First, although Spi‐B−/− splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross‐linking. Secondly, Spi‐B−/− mice display abnormal T‐dependent antigenic responses in vivo and produce low levels of antigen‐specific IgG 1, IgG 2a and IgG 2b after immunization. Finally, Spi‐B−/− mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild‐type animals, germinal centers in Spi‐B−/− mice are smaller and short‐lived with significantly increased numbers of apoptotic B cells. Taken together, these results demonstrate that Spi‐B is essential for antigen‐dependent expansion of B cells, T‐dependent immune responses and maturation of normal germinal centers in vivo.