The prevailing paradigm is that T cell differentiation is dependent on interactions between stem cells and neuroectodermal thymic cells in the context of a three-dimensional environment. We evaluated the utility of a three-dimensional matrix, the Cytomatrix®, to facilitate T cell differentiation. Thymus stroma grown on the Cytomatrix and seeded with hematopoietic progenitors was observed to support the development of both CD4+ and CD8+ T cells. Murine transgenic models used to address T cell selection demonstrated that both positive and negative selection was maintained in the context of MHC Class I. These data demonstrate that this in vitro system using neuroectoderm tissue is capable of the efficient production of T cells from hematopoietic progenitors and presents the possibility of generating and adoptively transferring immune cells to patients.