In the thymus, T cell development proceeds by successive steps of differentiation, expansion, and selection. Control of thymocyte proliferation is critical to insure the full function of the immune system and to prevent T cells from transformation. Deletion of the cell cycle inhibitor p16 INK4a is frequently observed in human T cell neoplasias and, in mice, gene targeted inactivation of the Ink4a locus enhances thymocyte expansion and predisposes mutant animal to tumorigenesis. Here, we investigate the mechanism by which p16 Ink4a controls thymocyte development by analyzing transgenic mice expressing the human p16 INK4a into the T cell lineage. We show that forced expression of p16 INK4a in thymocytes blocked T cell differentiation at the early CD4− CD8− CD3− CD25+ stage without significantly affecting the development of γδ T cells. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombinase activating gene (RAG)-2-deficient mice (RAG-2−/−). Upon anti-CD3ε treatment in vivo, p16 INK4a-expressing RAG-2−/− thymocytes were not rescued from apoptosis, nor could they differentiate. Our data demonstrate that expression of p16 INK4a prevents the pre-TCR-mediated expansion and/or survival of differentiating thymocytes.