The generation of diverse B and T cell antigen receptor repertoires during early lymphocyte development is dependent on the gene recombination activity of the Rag proteins. As B and T lymphocytes develop, the balance between proliferation and differentiation, along with the expression of Rag during specific parts of the cell cycle, must be tightly regulated to maintain genomic integrity and ensure the production of diverse pools of B and T cells. In this study, we used Abelson murine leukemia virus transformed B cell lines to identify novel pathways and factors responsible for the repression of Rag transcription at a specific point in B cell development. We demonstrated that the transcription factor early B cell factor 1 (Ebf1) and c-Myb, positive regulators of Rag transcription during early B cell development, function as repressors of baseline Rag expression at this later stage. In this context, Ebf1 and c-Myb act downstream of the signaling molecule Stat5 to repress differentiation and promote proliferation and survival of transformed cells. Ebf1 and c-Myb represent attractive therapeutic targets in hematopoietic malignancies such as acute lymphoblastic leukemia.