T cell differentiation in human marrow was studied in Dexter type long- term bone marrow cultures. In these cultures, T lymphocyte colony- forming units (TL-CFU), E rosette-forming cells (E+), and T3+, T4+, and T8+ cells (assayed by indirect immunofluorescence) were found to be present for at least 7 weeks. It was investigated whether the existence of T cells in long-term culture resulted from the persistence of inoculated T lymphocytes or from the production by immature progenitors. No significant numbers of E+, T3+, T4+, or T8+ cells were detected in cultures that were established from E+ lymphocyte-depleted bone marrow, indicating little or no production of T lymphocytes from E- negative precursors. On the other hand, bone marrow cells purged of E+ lymphocytes did not contain TL-CFU, but appeared to regain high numbers of TL-CFU during Dexter culture; this suggested that an earlier step in T cell differentiation may take place in this culture system. The generation of TL-CFU in the E-negative long-term marrow cultures only occurred when an adherent stroma layer had been established in the culture flask; it did not require added mitogens or detectable interleukin 2 in the culture medium. TL-CFU in fresh marrow (TL-CFU II) are mature (E+, T3+) T cells and are capable of producing helper (T4+) and suppressor/cytotoxic (T8+) phenotype cells in colonies. The TL-CFU newly formed in E-depleted Dexter cultures (TL-CFU I) are distinct from this population, as they are E-negative and give rise to colonies of the helper type only. T3 cell depletion of the marrow inoculum prior to culture did not prevent the appearance of TL-CFU I in long-term culture; this suggests that TL-CFU I are derived from an E- and T3- precursor (pre-TL-CFU).