CD34⁺ cells initiating hematopoiesis in the cytokine-supplemented (pre-CFU) assay express the 103B2/9E10 and 105.A5 epitopes. BMMNC were sorted into (□) CD34⁺, (⧫) CD34⁺ 103B2/9E10⁺, (○) CD34⁺103B2/9E10^lo/− (A and C) or (□) CD34⁺, (⧫) CD34⁺105.A5⁺, (○) CD34⁺ 105.A5^lo/− subpopulations (B and D) (as described in Materials and Methods) and assayed for their ability to initiate and maintain hematopoiesis in a stroma-independent, cytokine-supplemented culture. Cultures were established in triplicate using 1 × 10³ sorted cells per well in medium supplemented with 10 ng/mL each of purified recombinant human IL-1β, IL-3, IL-6, G-CSF, GM-CSF, and SCF. Additional factors were added at the same concentrations on days 7, 14, and 21. On days 7, 14, and 21, the cells were harvested, washed, and assayed for nucleated cell number and CFU-GM as previously described. The results are expressed as the mean number ± SE of nucleated cell number (A and B) and CFU-GM (C and D) recovered at days 7, 14, 21, and 28 for each group. A representative experiment (1 of 3) is shown.