Developmental stage-, subset-, and lineage-specific CD8 enhancers have been identified recently by transgenic reporter analyses. Enhancer E8(II) (CIV-4,5) is active in both immature double-positive thymocytes (DP) and mature CD8 single-positive (SP) thymocytes and T cells, whereas E8(I) (CIII-1,2) directs expression only in mature cells. In mice lacking either E8(I) (CIII-1,2) or E8(II) (CIV-4,5), there was no effect on CD8 expression in DP thymocytes. However, deletion of both enhancers resulted in variegated expression of CD8, with appearance of CD4(+)CD8(-) SP thymocytes expressing surface markers characteristic of DP thymocytes. Consequently, fewer mature CD8(+) T cells developed from the reduced pool of DP cells. These results suggest that the initiation of CD8 expression is mediated by cis-regulatory elements that are distinct from any that may be involved in maintenance of expression.