The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.