PU.1+/-Spi-B-/- mice exhibit reduced numbers of immature and mature B lymphocytes, which exhibit severe defects in response to BCR-mediated stimulation and poor survival. We found that expression of c-rel, a member of the Rel/NF-κB family, is dramatically reduced in PU.1+/-Spi-B-/- splenic B cells. Analysis of the murine c-rel promoter identified three PU.1/Spi-B binding sites critical for c-rel promoter activity. Furthermore, reintroduction of Rel protein restored wild-type B cell numbers to mice reconstituted with PU.1+/-Spi-B-/- bone marrow. These findings are the first to demonstrate that a member of the Rel/NF-κB family is directly regulated by Ets proteins and dissect the molecular basis for the function of two Ets factors, PU.1 and Spi-B, in promoting B lymphocyte survival.