Medullary thymic epithelial cells (mTECs) are specialized for inducing central immunological tolerance to self-antigens. To accomplish this, mTECs must adopt a mature phenotype characterized by expression of the autoimmune regulator Aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. The mechanisms that control mature Aire(+) mTEC development in the postnatal thymus remain poorly understood. We demonstrate here that, although either CD4(+) or CD8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mTEC population requires autoantigen-specific interactions between positively selected CD4(+) thymocytes bearing autoreactive T cell receptor (TCR) and mTECs displaying cognate self-peptide-MHC class II complexes. These interactions also involve the engagement of CD40 on mTECs by CD40L induced on the positively selected CD4(+) thymocytes. This antigen-specific TCR-MHC class II-mediated crosstalk between CD4(+) thymocytes and mTECs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mTEC population competent for ensuring central T cell tolerance.