A small population of human marrow cells has been shown to be differentiated in vitro by thymic extract into cells bearing T-lymphocyte (thymus-derived lymphocyte) characteristics. By a similar method, the differentiation of human peripheral blood lymphocytes has been studied. A discontinuous gradient of bovine serum albumin was used to isolate lymphocytes into four layers and cells from layers I and III demonstrated the greatest potential for differentiation by human thymic extract. Appearance of T-lymphocyte characteristics was recognized by the spontaneous E-rosette technique with sheep erythrocytes. Ability of human marrow cells to be differentiated under the influence of human thymic extract was abolished by specific inhibitors of nucleic acid synthesis, however, had no inhibitory effect on the maturation of peripheral blood lymphocytes during a 2 hr incubation with human thymic extract but puromycin, an inhibitor of protein synthesis, abolished this differentiative step in cells of layer I. It is suggested from these studies that many of the cells in peripheral blood that are differentiable by thymic extract are at a stage of maturation more advanced than those in human marrow that are also differentiable by thymic extract.