Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, and cancer-germline genes. Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes. These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression.