The thymus is mainly composed of two types of epithelial cells, medullary thymic epithelial cells and cortex thymic epithelial cells (mTECs and cTECs). The tissue structure and mechanism for T cell development are complicated, with generation of the thymus regulated by complex molecular and cellular interactions of the thymic microenvironment during embryogenesis. Since the development of organ regeneration techniques became available, complete in vitro regeneration of the thymus has been attempted. Steric induction systems are thought to be optimal for tissue regeneration, but three-dimensional (3-D) induction of TECs from induced pluripotent stem cells (iPSCs) has not yet been reported. Here, we demonstrate the induction of functional TECs from iPSCs by a 3-D spheroid culture system with recruitment of robust numbers of T cells into the peripheral blood. Purified iPSC-derived TECs showed a sufficient expression level of FoxN1 comparable to TECs, and phenotypic analysis revealed that iPSC-derived TECs were expressing K5. Moreover, transplants of cell aggregations into recipient mice were not rejected and there was normal support of T cell development. Functional analysis revealed that T cells showed immune tolerance to both donor and recipient MHCs and could reject an allogeneic third party's skin graft without tumorigenesis. Taken together, these findings raised the possibility of using iPSC-derived TECs induced by 3-D spheroid culture in future regenerative therapy for patients with immunodeficiency.