CD4 and CD8 are crucial for the development and function of T cells. An intergenic deoxyribonuclease I hypersensitive site region (cluster CIII) directs expression in mature CD8 T cells only. Here, we show that two further independent regions from the CD8 gene locus in conjunction with cluster CIII restore transgene expression in appropriate immature thymocytes. Deletion of two of the intergenic cluster CIII DNasel-HSS in homozygous mutant mice affects expression of CD8αα homodimers on intraepithelial T cells (IEL), particularly on the γδTCR + subset. Surprisingly, none of the thymocyte or peripheral αβTCR T cell subsets are affected by this mutation, indicating hierarchical activation of these elements within the different T cell subsets.