Following positive selection, thymocytes migrate into the medulla where they encounter diverse tissue-restricted antigens (TRAs) that induce self-tolerance. Both medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present TRAs to induce negative selection of autoreactive thymocytes, but their relative contribution remains unresolved. Previous studies using genetic models to impair antigen presentation by mTECs or DCs demonstrated both cell types could induce negative selection.