Control of CD8alpha transcription during development of alpha/beta T cell receptor (TCR) T lymphocytes is mediated by at least two distinct stage-specific cis-acting transcriptional mechanisms (i.e., enhancers). On the CD8alpha(-/-) knockout (KO) background, cis-mechanism I and cis-mechanism II together mediate appropriate stage- and sublineage-specific transgenic (Tg) CD8alpha expression and "rescue" development of peripheral CD8(+) single-positive (SP) cytotoxic T lymphocytes (CTLs). In contrast, on the wild-type (WT)/CD8(+/+) or CD8alpha(-/-)KO backgrounds, a CD8alpha Tg directed by cis-mechanism I alone is activated during the double negative [DN] to double positive [DP] transition and expressed up to the CD3(low/intermediate) DP stage but not in more mature DP or SP thymocytes or peripheral T cells. As loss of cis mechanism I activity occurs around the onset of positive selection, it is possible that events associated with TCR/major histocompatibility complex (MHC) interactions and selection are involved in initiating these changes in CD8alpha transcription. To examine this issue, phenotypic and functional studies were performed for thymocytes and T cells of CD8alpha(-/-) KO mice that expressed a CD8alpha Tg under control of cis-mechanism I only. Despite loss of CD8alpha expression at the DP CD3(low/intermediate) stage, increased populations of mature CD3(hi)CD4(-)CD8(-) thymocytes and CD3(+)CD4(-)CD8(-) peripheral T cells were detected. By several criteria, including MHC class I-restricted antigen recognition, these cells have at least partially undergone positive and negative selection. Therefore, initiation of selection and sublineage commitment are determined before loss of cis-mechanism I-mediated control of CD8alpha transcription. Further, CD8 expression beyond the CD3(low/intermediate) DP thymic stage is not essential for CTL development in vivo or function.