OBJECTIVE The effect of stem cell factor (SCF), flt3-ligand (FL), and interleukin (IL)-3 (SF3) in combination with hepatocyte growth factor (HGF), thrombopoietin (TPO), and Hyper-IL-6 on maintenance and differentiation of early human peripheral blood-derived progenitor cells was investigated. METHODS Single sorted CD34(+) 38(-) cells were cultured with various combinations of these growth factors in order to identify the most effective cytokine combination. Then, lineage-depleted cells were stimulated for 7 d in bulk culture before they were assessed by flow cytometry and in functional assays. RESULTS The highest number of clones in the single-cell assay was obtained after culture with SF3 + TPO + HGF. Cell expansion with SF3 + TPO + HGF yielded an increase of the total cell number (11-fold), the number of CD34(+) cells (sevenfold), colony forming cells (CFC; 13-fold), granulocytes (CD15/66b(+); 45-fold) and B-cells (CD19/20(+); 55-fold). However, the number of long-term culture initiating cells (LTC-IC) decreased from 779 +/- 338 per 1 x 10(5) CD34(+) cells on day 0 to 253 +/- 115 on day 7. In parallel, the number of pluripotent mouse repopulating cells decreased by the factor 11, and no significant change in the proportion of human myeloid or lymphoid cells found in the mouse bone marrow was noted. CONCLUSIONS The observation that mature cells of different lineages are generated and that transplantable multipotent hematopoietic cells are lost during culture suggests the differentiation of early hematopoietic progenitors toward lineage committed cells by the tested cytokines. The detection of cells expressing B-lymphoid markers after culture indicates a possible role in the propagation of B-cells.