Proliferation and differentiation in AFT024 cultures depends on addition of defined cytokines. CD34⁺/Lin⁻/DR⁻ cells (1,000 cells/well) were cultured on AFT024 in 96-well plates with the cytokine combinations indicated. Cytokines were added only once at culture initiation and weekly half media changes contained fresh IL-2 alone. Abbreviations for cytokines in all figures are as follows: 7, IL-7; F, FL (Flt3 ligand); K, KL (c-kit ligand); 2, IL-2; 3, IL-3. After 28 to 35 days in culture, 42 wells per condition from cells derived from three donors were analyzed for proliferation and presence or absence of NK and B-lineage cells. (A) Relative proliferation was determined by flow cytometry as the number of events analyzed per minute. Data are the mean ± SEM for replicate wells analyzed in parallel with the total contents of a harvested well analyzed in a constant volume of approximately 180 μL. The P values shown are comparisons between adjacent cytokine combinations. (B) The percentage of positive wells (of 42 wells analyzed per condition) is shown for NK cells (black bars) and B-lineage cells (hatched bars) for each cytokine combination.