We know little about the way mature CD4 (helper) and CD8 (killer) T cells develop from thymic CD4+CD8+ precursors. Here we show that small but not large CD4+CD8+ cells with high levels of the alpha beta T cell receptor (TcRhigh) result from positive selection. Neither CD4+CD8+ cells with low TcR levels nor large CD4+CD8+ thymocytes with high TcR levels differentiate in vitro. However, small CD4+CD8+ cells with high TcR levels develop in vitro into mature cells by gradually decreasing the surface levels of one or the other co-receptor and acquiring the potential to respond with proliferation to ligation of the TcR. Small CD4+CD8+ cells with high levels of a major histocompatibility complex (MHC) class I-restricted transgenic TcR develop in vitro exclusively into CD4-CD8+ cells while small CD4+CD8+ TcRhigh cells with heterogeneous TcR from various mice yield both CD4 and CD8 T cells. While these experiments are consistent with an instructive model of CD4/CD8 lineage commitment they do not rule out other mechanisms which require multiple TcR-MHC ligand interactions in the generation of mature alpha beta T cells.