Many mammalian genes, including those encoding antigen receptors, contain more than one enhancer element. Deleting one element often does not prevent expression, but functional redundancy has never been directly demonstrated by gene targeting of multiple elements. We demonstrate that simultaneous deletion of two enhancer/LCR-like elements in the TCR Cγ1 cluster, HsA and 3′E_Cγ1, severely diminishes TCRγ transcription, selectively impairs development of γδ thymocyte subsets, but only modestly reduces TCRγ gene rearrangement, while deletion of each element separately has little effect. In contrast to these results in thymocytes, deletion of HsA alone reduces transcription of one Vγ gene specifically in peripheral γδ T cells. Thus, the two elements exhibit functional redundancy in thymocytes but also have unique functions in other settings.