Significance Aire controls immunological tolerance by driving promiscuous expression of a large swath of the genome in medullary thymic epithelial cells (mTECs). Its molecular mechanism remains enigmatic. To address the hypothesis that Aire impacts chromatin organization through widespread promotion of superenhancer–promoter loops, we performed genome-wide, high-resolution chromosome-conformation capture (Hi-C) experiments on ex vivo mTECs from thymi of mice expressing Aire or not. Integration of the resulting data with inventories of the genome-wide locations of Aire, subunits of the mediator and cohesin complexes, and the chromatin-domain enforcer, CCCTC-binding factor (CTCF), indeed argued for a looping model, which was further supported by biochemical and in vivo loss-of-function analyses.