Signaling pathways such as the pre-TCR and Wnt pathways regulate alpha/beta T cell differentiation in thymus. Mice lacking an essential component of the pre-TCR exhibit arrest at the (CD4(-)CD8(-)) (CD44(-)CD25(+)) stage (DN3) of thymocyte development, and introduction of p53 deficiency into those mice abrogates this arrest, resulting in transition to the (CD4(+)CD8(+)) double-positive (DP) stage. This paper examines the effect of inactivation of p53 on thymocyte development in Bcl11b(-/-) mice that exhibit arrest at the DN3 or (CD4(-)CD8(+)) immature single-positive (ISP) stage. No DP thymocytes were detected in thymocytes of adoptive transfer experiments in scid mice that were derived from p53(-/-)Bcl11b(-/-) precursors but ISP thymocytes increased in the proportion and in the cell number approximately three times higher than those from Bcl11b(-/-) precursors. Consistently, the level of apoptosis decreased to the level of wild-type precursors. These results suggest that inactivation of p53 is sufficient for DN3 thymocytes to differentiate into the ISP, but not to DP, stage of thymocyte development in Bcl11b(-/-) mice. This provides evidence for a novel p53-mediated checkpoint that regulates the transition from the DN3 to ISP stage of thymocyte development.