Medullary thymic epithelial cells (mTECs) are a heterogeneous population in terms of the spectrum of tissue-restricted Ags (TRAs) expressed from each cell for ensuring the elimination of autoreactive T-cells. Additionally, mTECs comprise cells at different developmental stages and/or in various activation conditions. Because of these heterogeneities, it is unclear whether mTECs are composed of any particular subsets possessing unique properties for their developmental pathway and/or immunological function. Here, we report a distinct mTEC subset characterised by expression of Ly6 family protein prior to and concomitant with Aire expression during its differentiation. Ly6C/6G+ mTECs, constituting 5%–15% of mature mTECs, were preferentially localised at the cortico-medullary junction, and expressed high levels of TRAs and thymocyte-attracting chemokines. Remarkably, Ly6C/6G+ mTECs were absent in Aire-deficient mice, suggesting that this subset requires Aire and/or Aire+ mTECs for its production. Uniquely, Ly6C/6G+ mTECs lack a post-Aire stage because of a tendency to die after Aire had been expressed. With a TCR-transgenic model in mice, we found that in vivo depletion of Ly6C/6G+ mTECs frequently induced organ-specific autoimmunity. We suggest that Ly6C/6G+ mTECs serve as an important source of TRAs for efficient cross-presentation during establishment of self-tolerance.