Abstract The autoimmune regulator (Aire) protein, a key transcriptional regulator expressed in medullary thymic epithelial cells (mTECs), is crucial for central tolerance. Aire is also found in extrathymic Aire-expressing cells (eTACs) in secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells that are capable of inducing immune tolerance, but the precise identity of eTACs has remained unclear. To define eTACs, we utilized a combination of single-cell multiomics, transgenic murine models, and functional approaches. We found that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells and an Aire-high population co-expressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we termed Janus cells (JCs). eTACs, particularly JCs, have highly accessible chromatin and share remarkable homology with mTECs. Additionally, transgenic self-antigen expression by eTACs is sufficient to induce negative selection of cognate autoreactive T cells and prevent autoimmune diabetes. To better define the function of extrathymic Aire in eTACs and its contribution to peripheral immune tolerance, we have generated a Peripheral Aire KnockOut (PAKO) mouse (Vav1-Cre x Aire-fl/fl). Using intracellular Aire staining by flow cytometry, we validated that PAKO mice lack all eTACs in secondary lymphoid organs while maintaining normal thymic Aire expression in mTECs. Studying the biology of eTACs and peripheral Aire will help further elucidate the function of Aire and define basic peripheral tolerance mechanisms, which may have significance for a range of clinical applications from autoimmunity to tumor immunity to maternal-fetal tolerance. Supported by ARCS Foundation