The transcription factor recombination signal binding protein‐J (RBP‐J) functions immediately downstream of the cell surface receptor Notch and mediates transcriptional activation by the intracellular domain of all four kinds of Notch receptors. To investigate the function of RBP‐J, we introduced loxP sites on both sides of the RBP‐J exons encoding its DNA binding domain. Mice bearing the loxP‐flanked RBP‐J alleles, RBP‐J^f/f, were mated with Mx‐Cre transgenic mice and deletional mutation of the RBP‐J gene in adult mice was induced by injection of the IFN‐α inducer poly(I)–poly(C). Here we show that inactivation of RBP‐J in bone marrow resulted in a block of T cell development at the earliest stage and increase of B cell development in the thymus. Lymphoid progenitors deficient in RBP‐J differentiate into B but not T cells when cultured in 2′‐deoxyguanosine‐treated fetal thymic lobes by hanging‐drop fetal thymus organ culture. Competitive repopulation assay also revealed cell autonomous deficiency of T cell development from bone marrow of RBP‐J knockout mouse. Myeloid and B lineage differentiation appears normal in the bone marrow of RBP‐J‐inactivated mice. These results suggest that RBP‐J, probably by mediating Notch signaling, controls T versus B cell fate decision in lymphoid progenitors.