Dietary vitamin B6 (pyridoxine) deficiency in young Lewis rats results in a reduction of T lymphocyte numbers and defects of cellular immunocompetence. In vitro studies of thymic epithelial (TE) cells, responsible for inducing T lymphocyte differentiation, revealed that maintenance on a vitamin B6 deficient diet for 2 weeks resulted in a severe defect in TE cell function. When the deficient animals were returned to a normal diet, TE cell function was restored. Exposure of lymphoid precursors from neonatally thymectomized or vitamin B6-deficient donors to normal TE monolayers resulted in their conversion to functional T lymphocytes, as measured by their response in MLR and to mitogens. However, TE monolayers from vitamin B6-deficient animals were unable to effect such a maturation of T lymphocytes. Therefore, it is suggested that the defect in cellular immunocompetence following this dietary deficiency is due, at least in part, to the inability of TE cells to effect the differentiation of T lymphocyte precursors to functional T lymphocytes. The dietary deficiency does not, however, impair lymphoid precursors, which can be stimulated to further differentiation by exposure to normal TE cell monolayers.