Because the deletion of self-reactive T cells is incomplete, thymic development of natural Foxp3+CD4+ regulatory T cells (Treg cells) is required for preventing autoimmunity. However, the function of T cell antigen receptor (TCR) specificity in thymic Treg cell development remains controversial. To address this issue, we generated a transgenic line expressing a naturally occurring Treg cell-derived TCR. Unexpectedly, we found that efficient thymic Treg cell development occurred only when the antigen-specific Treg cell precursors were present at low clonal frequency (<1%) in a normal thymus. Using retroviral vectors and bone marrow chimeras, we observed similar activity with two other Treg cell-derived TCRs. Our data demonstrate that thymic Treg cell development is a 'TCR-instructive' process involving a niche that can be saturable at much lower clonal frequencies than is the niche for positive selection.