Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8 III enhancer, which directs expression in double-positive (DP) thymocytes, and E8 II, which is active in DP cells and CD8+ T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8 III enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8 III in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8 II is also deleted. These results indicate that E8 III is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development.