Immunosuppressive drugs and cytotoxic chemotherapy agents are designed to kill or suppress autoreactive, alloaggressive, or hyperinflammatory T cells, or disseminated malignancies. However, they also cause severe immunological side effects ranging from interrupted thymopoiesis and general immunodeficiency to, paradoxically, autoimmunity. Consistent with the cross-talk between thymocytes and stromal cells, we now show that these common therapeutic agents have major effects on murine thymic epithelial cells (TEC), crucially required to rebuild immunity posttreatment. We show that the immunosuppressant cyclosporine A, which has been linked to a thymus-dependent autoimmune syndrome in some patients, causes extensive loss of autoimmune regulator (Aire+) tolerance-inducing MHC class II high medullary TEC (mTEC high). Post-cyclosporine A, Aire expression was restored within 7 days. Full recovery of the mTEC high subset occurred within 10 days and was linked to a decrease in a relatively resistant MHC class II low mTEC subset (mTEC low), consistent with a previously described precursor-product relationship. Cyclophosphamide and dexamethasone caused more extensive ablation of thymocytes and stromal cells but again severely depleted tolerance-inducing mTEC high. Together, these data show that Aire+ mTECs are highly sensitive to damage and that mTEC regeneration follows a conserved pattern regardless of the treatment regimen used.