Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib-/-Spic+/-). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib-/- mouse spleens. Spib-/-Spic+/- B cells had restored proliferation compared with Spib-/- B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib-/-Spic+/- phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib-/-Spic+/- B cells compared with Spib-/- B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.