Two candidate genes for controlling thymocyte differentiation,T-cellfactor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins^1,2. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally³. Here we generate two independent germline mutations in ^Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8⁺, immature single-positive to the CD4⁺/CD8⁺ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.