Regulating RAG activity in B lineage cells is crucial to prevent deleterious events that can be caused by the presence of DNA double strand breaks. To identify negative regulators of RAG expression, we conducted an unbiased cDNA library screen in Abelson murine leukemia-virus transformed pro-B cells. We found that overexpression of the transcriptional repressor Gfi1b downregulates RAG expression in pro-B cell lines and primary B lineage cells from bone marrow. Gfi1b binds directly to a region of the RAG locus upstream of the B-cell specific Erag enhancer and its activity depends on its association with chromatin modifying cofactors. In addition, Gfi1b's effect on RAG levels appears to be mediated in part by repression of FoxO1, a recently identified positive regulator of RAG expression. Gfi1b-deficient cell lines exhibit increased RAG levels as well as an increase in the overall number of DNA double strand breaks per cell when compared to their wildtype counterparts, suggesting that Gfi1B may be critical to maintain genome integrity. Moreover, microarray experiments revealed that Gfi1b controls the expression of a suite of B lineage-specific genes, including the immunoglobulin kappa locus and the transcription factor SpiB. We identify Gfi1B as a novel regulator of RAG expression that may also be involved in the execution of genetic programs that govern B cell development.