Autoimmune regulator (Aire) has a unique expression pattern in thymic medullary epithelial cells (mTECs), in which it plays a critical role in the activation of tissue‐specific antigens. The expression of Aire in mTECs is activated by receptor activator of nuclear factor κB (RANK) signaling; however, the molecular mechanism behind this activation is unknown. Here, we characterize a conserved noncoding sequence 1 (CNS1) containing two NF‐κB binding sites upstream of the Aire coding region. We show that CNS1‐deficient mice lack thymic expression of Aire and share several features of Aire‐knockout mice, including downregulation of Aire‐dependent genes, impaired terminal differentiation of the mTEC population, and reduced production of thymic Treg cells. In addition, we show that CNS1 is indispensable for RANK‐induced Aire expression and that CNS1 is activated by NF‐κB pathway complexes containing RelA. Together, our results indicate that CNS1 is a critical link between RANK signaling, NF‐κB activation, and thymic expression of Aire.