The aim of the study is to define the ability of Flt3 ligand, MGDF, Epo and G-CSF to modulate the expansion of different hematopoietic compartments in association with a basic cocktail of SCF + IL-3 + IL-6 (S36). CD34+ cells from normal bone marrow were cultured in stroma-free, serum-free medium for 10 days. Using various concentrations of cytokines, total cells could be expanded up to 5200-fold, CD34+ cells up to 78-fold, CFU-GM up to 143-fold, BFU-E up to 46-fold, CFU-MK up to six-fold and LTC-IC up to four-fold. The results were assessed by multiparametric analysis of variance. Three factors had a significant stimulatory effect on the late precursor compartment: Epo (P < 10(-5)), G-CSF (P=5 x 10(-3)) and FL (P=10(-5)). Two were critical for CD34+ cell expansion: FL (P=4 x 10(-5)) and Epo (P=6 x 10(-5)), while two were critical for BFU-E expansion: MGDF (P=8 x 10(-4)) and FL (P=0.017). FL strongly stimulated CFU-GM expansion (P < 10(-5)), whereas none of the growth factors studied had any effect on CFU-MK. FL (P=10(-4)) and MGDF (P=0.002) were essential to obtain high levels of expansion of LTC-IC as determined in limiting dilution assays. In the light of the above results showing a preferential effect on the expansion of precursor cells (3080-fold), CD34+ cells (53-fold), CFU-GM (134-fold), BFU-E (46-fold) and LTC-IC (five-fold), the combination SCF, IL-3, IL-6, FL, MGDF, Epo and G-CSF was chosen as a putative cytokine cocktail for further studies on long-term culture. Sustained production of precursor cells, progenitor cells, LTC-IC and E-LTC-IC for up to 100 days reflects the persistence of very primitive stem cells. This suggests that these populations are probably able to undergo self-renewal divisions. The above combination of cytokines meets the required criterion for potential clinical application, which may be defined as an effective capacity to expand all cell compartments, using as the starting material high concentrations of low purity CD34+ cells.