Twenty thousand CB CD34⁺ cells were resuspended in 1 mL IMDM containing 10% pooled human serum and cultured for up to 8 weeks in 24-well plates. In the first series (demidepopulation wells) cells in 1 mL of a single well were demidepopulated every week by removal of one half the culture medium (and cells), which was replaced with the same volume of fresh medium and growth factors. In the second series (expansion wells), 2 × 10⁴ CD34⁺ cells were prepared as described above in 24-well plates in 1 mL. Every week the cells were resuspended in twice the volume of culture medium, so that every week additional new 24-wells were set up. In the third series (flasks) 2 × 10⁴ CD34⁺ cells/mL were layered on the bottom of T flasks and every week the culture volume was doubled. At the indicated time points, the cells were harvested, counted, and aliquots analyzed for CD34⁺ antigen expression. Suitable aliquots were assayed in semisolid assays for CFC and on stroma cocultures for LTC-1C enumeration. Mean ± SEM of 4 to 6 separate experiments performed in quadruplicate.