The developmental and subset-specific expression of the CD8 genes is under the control of a complex array of regulatory elements distributed along the locus and characterized by DNaseI hypersensitivity. Here we describe the phenotype of mice in which hypersensitive sites 1 and 2 (HSS1 and 2) of DNaseI hypersensitive Cluster II (CII), which are located upstream of the CD8α gene, were deleted by targeted homologous recombination of the endogenous locus. Knockout mice exhibit a variegated expression of the CD8α gene, particularly among the immature CD4⁺8⁺ TCR^lo thymocyte population. We propose that HSS Cluster II regulatory elements are essential in ensuring initiation of chromatin remodeling and establishment of an open configuration in all developing thymocytes that undergo the double-negative to double-positive transition. Furthermore, these sequences contribute to the levels of expression of the CD8α gene.