Thymocytes with a CD4(hi)CD8(lo) coreceptor-skewed (CRS) phenotype have been shown to contain precursors for CD8 single-positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4(hi)CD8(lo) cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I-restricted P14 T cell receptor (TCR), on the H-2bm13 background, generate a large number of CD4(hi)CD8(lo) CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4(hi)CD8(lo) population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up-regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re-expressed CD8 but failed to re-express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide-pulsed antigen-presenting cells. Our data suggest that CD4(hi)CD8(lo) CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.