The process of thymocyte development culminates in the maturation of helper (CD4+) and cytotoxic (CD8+) T cells from their common precursors, the CD4+ CD8+ double-positive cells 1, 2. A crucial step during lineage specification is the termination of expression of either the CD4 or the CD8 coreceptor 3, 4. A silencer element within the first intron of the CD4 gene is sufficient for CD4 transcriptional repression in cells of the cytotoxic lineage, as well as in thymocytes at earlier stages of differentiation 5, 6. Here we show that the function of the CD4 silencer is required only at distinct stages of development. Its deletion before the initiation of lineage specification resulted in CD4 derepression throughout thymocyte differentiation. By contrast, once cells committed to the cytotoxic CD8+ lineage, the CD4 locus remained silent through subsequent mitoses, even when the silencer element was excised. The epigenetic inheritance of the silenced CD4 locus was not affected by the inhibition of DNA methylation or histone deacetylation, and may thus involve other mechanisms that ensure a stable state of gene expression.