Thymocyte and thymic epithelial cell (TEC) development are interdependent processes. Although lineage relationships among progressively maturing thymocyte subsets have been characterized, the developmental relationships among TEC subsets are obscure. Because epithelial cells express distinct keratin (K) species as a function of differentiation stage and proliferative status, we used K expression patterns to identify mouse TEC subsets and determine their lineage relationships. As expected, cortical and medullary TEC subsets express distinct K expression patterns in the normal thymus. However, we detected two distinct cortical TEC subsets, a major K8⁺K5⁻ subset and a minor K8⁺K5⁺ subset, which is highly represented at the cortico-medullary junction. Both cortical TEC subsets are also present in recombination activating gene 1 (RAG-1^−/−) and TCRβxδ^−/− thymi in which T-cell development is blocked at the CD4⁻CD8⁻CD25⁺CD44⁻ pre-T cell stage. In contrast, K8⁺K5⁺ TECs predominate in the thymi of human CD3ɛ transgenic mice in which thymocyte development is blocked at an earlier CD4⁻CD8⁻CD25⁻CD44⁺ stage. Transplantation of newborn human CD3ɛ transgenic thymi under the kidney capsule of RAG-1^−/− mice results in the emergence of K8⁺K5⁻ TECs concomitant with the appearance of CD25⁺ thymocytes. Together, the data suggest that cortical TEC development proceeds from a K8⁺K5⁺ precursor subset to a K8⁺K5⁻ stage in a differentiation process concomitant with T-cell lineage commitment.