The thymic microenvironment provides essential support for the generation of a functional and diverse population of human T cells. In particular, the three-dimensional (3D) thymic architecture contributes to critical cell-cell interactions. We report that thymic stroma, arrayed on a synthetic 3D matrix, supports the development of functional human T cells from hematopoietic precursor cells. Newly generated T cells contain T-cell receptor excision circles and are both fully mature and functional. The coculture of T-cell progenitors with thymic stroma can thus be used to generate de novo functional and diverse T-cell populations. This novel tissue engineered thymic system has biological applications for the study of T-lymphopoiesis and self-tolerance as well as potential therapeutic applications including the immune reconstitution of immunocompromised patients and the induction of tolerance in individuals receiving tissue or organ transplants.