Differentiation of MHC class II-selected thymocytes toward the CD4(+) helper lineage depends on function of the transcription factor ThPOK, whose expression is repressed in CD8(+) cytotoxic lineage cells by a transcriptional silencer activity within the distal regulatory element (DRE) in the Thpok gene. Interestingly, the DRE also functions as a transcriptional enhancer. However, how the DRE exerts such dual functionality remains obscure. In this study, we dissected the DRE and identified DNA sequences specifically responsible for enhancer activity, and designated this as the thymic enhancer. Removal of the thymic enhancer from the murine Thpok locus resulted in inefficient ThPOK induction, thereby inducing a redirection toward alternative CD8(+) cytotoxic lineage in a proportion of MHC class II-selected cells, even when they express monoclonal MHC class II-restricted transgenic TCR. Thus, regulation of contiguous but separable sequences with opposite function in the DRE plays an important role in precise coupling of TCR signaling with the selection process of two opposite lineages.