Cortical CD4+CD8+ thymocytes mature into CD4+ or CD8+ thymocytes through a process termed positive selection. To better define differentiative stages of CD4+CD8+ thymocyte development in positive selection, we performed a phenotypic analysis of CD4+CD8+ thymocytes from H-Y mice mated to various genetic backgrounds. We have previously shown that coordinate binding of the H-Y TCR and the CD8 coreceptor to the restricting Db MHC class I molecule is required for the efficient positive selection of this TCR. In this study we have used TCR, CD5, and CD45 expression levels as markers for thymocyte maturation. Lack of CD8/Db interaction was achieved by introducing a mutation that abrogates CD8 binding in the alpha 3 domain of Db. We found that the absence of coreceptor ligation prevented TCR up-regulation in CD4+CD8+ thymocytes and resulted in a developmental arrest characterized by low levels of TCR and CD45. We have previously shown that deletion of CD4+CD8+ thymocytes expressing the H-Y TCR is facilitated by CD8 coreceptor ligation. Here we show that expression of the deleting ligand in the absence of coreceptor ligation caused CD5 up-regulation without concomitant TCR or CD45 up-regulation in CD4+CD8+ thymocytes. In a beta 2-microglobulin null background, introduction of the H-Y TCR caused the majority of CD4+CD8+ thymocytes to express an unusually low level of of the CD5 activation marker, suggesting that a low-affinity or noncognate TCR/MHC interaction may be required for initial CD5 up-regulation to intermediate levels. Collectively, these observations favor a maturational process in positive selection in which CD5 up-regulation precedes CD45 and TCR up-regulation.