Abstract Mice expressing a constitutively active form of STAT5b (Stat5b-CA mice) develop pre-B leukemia with low frequency (~1-2%). Microarray analysis of these leukemias shows decreased expression of genes involved in pre-BCR signaling. Stat5b-CA mice crossed to mice with defects in a coherent signaling pathway downstream of the pre-BCR involving BLNK, BTK, or PKCβ developed pre-B leukemia in > 75% of progeny. These leukemias exhibited reduced expression of a subset of NFκB target genes, which was important for transformation as Stat5b-CA x Nfkb1-/- mice also developed pre-B leukemia. Our results point to an unexpected role for NFκB1 as a tumor suppressor in pre-B cells. Microarrary analysis revealed that STAT5 activation resulted in reduced expression of several NFκB and FOXO target genes, thereby antagonizing these tumor suppressor pathways. To examine this regulation in further detail, we performed ChIP-seq analysis, which showed that STAT5 binds 38% of NFκB target genes that are expressed in pre-B cells. We also observed binding of STAT5 to several FOXO targets including: Foxo1, Cdkn1a, Cdkn2d, Blnk, Vegfa, Cxcr4, Bim, and Dgka. We have validated STAT5 binding to the FOXO targets Foxo1 and Cdkn2d and the NFκB targets Nfkb1, Rel, Irf4, Irf8, Ikzf1, and Ikzf3 by ChIP-qPCR. Our findings demonstrate that STAT5 activation antagonizes the pre-BCR-dependent NFκB and FOXO tumor suppressor pathways that limit pre-B cell proliferation and promote pre-B cell differentiation.