In several experimental systems analyzing the generation of single positive (SP) thymocytes from double positive (DP) thymocytes, CD4 SP cells have been shown to appear before CD8 SP cells. This apparent temporal asymmetry in the maturation of CD4 SP and CD8 SP thymocytes could either be due to divergent molecular differentiation programs of the two T cell lineages, or merely to slower degradation kinetics of the CD4 protein. To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells. We show that these TCRintCD69pos thymocytes represent a transitory stage in the mainstream alphabeta T cell lineage. The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells. Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.