CD4(+)Foxp3(+) Treg maintain peripheral tolerance and influence immune responses to foreign antigens. The thymus is an important source of Treg, but controversy exists as to whether T cells are selected into the Treg lineage based on signals received through TCR specific for self-peptides. To examine the specificity of TCR expressed by Treg and its effect on CD4(+) T-cell development, we generated Treg-TCR transgenic mice. Deletion of >90% of CD4(+) T cells in RAG-sufficient mice, and nearly 100% deletion in RAG(-/-) mice expressing this TCR indicate that the TCR is specific for an unknown, naturally expressed peptide in the thymus. Deletion occurs late in development, suggesting this peptide is presented by APC in the thymic medulla. These studies are the first to describe the effects of expressing a Treg-TCR on CD4(+) T-cell development. The implications of our data for models of Treg selection are discussed.