We have used gene-targeted mutation to assess the role of the T cell receptor δ (TCRδ) enhancer (Eδ) in αβ and γδ T cell development. Mice lacking Eδ exhibited no defects in αβ T cell development but had a severe reduction in thymic and peripheral γδ T cells and decreased VDJδ rearrangements. Simultaneous deletion of both Eδ and the TCRα enhancer (Eα) demonstrated that residual TCRδ rearrangements were not driven by Eα, implicating additional elements in TCRδ locus accessibility. Surprisingly, while deletion of Eδ severely impaired germline TCRδ expression in double- negative thymocytes, absence of Eδ did not affect expression of mature δ transcripts in γδ T cells. We conclude that Eδ has an important role in TCRδ locus regulation at early, but not late, stages of γδ T cell development.