Pretransplant and posttransplant use of hematopoietic growth factors in bone marrow transplantation (BMT) has shortened the time to engraftment. Severe neutropenia and thrombocytopenia have been the major clinical problems associated with autologous BMT. Efforts to maintain posttransplant levels of circulating neutrophils have focused on exploiting the synergistic action between various hematopoietic growth factor families. Ex vivo generation of distinct populations of expanded cells through simultaneous and sequential addition of hematopoietic growth factors was attempted. Cultures of CD34-selected cells with combinations of growth factors consisting of either recombinant human stem cell factor (rhSCF), recombinant human interleukin-6 (rhIL-6), and recombinant human interleukin-3 (rhIL-3) or rhSCF, rhIL-3, and recombinant human granulocyte-colony stimulating factor (rhG-CSF) generated two distinct but overlapping populations of cells. Delayed addition (on day 7) of rhIL-3 and rhIL-6 to cells cultured with rhSCF generated a population of cells significantly less mature than those cultured with continuous rhSCF, rhIL-3, and rhIL-6 alone. It appears that optimal generation of immediate and delayed cell populations can be achieved by simultaneous culture with rhSCF, rhIL-3, and rhG-CSF; and with rhSCF, rhIL-6, and rhIL-3. Questions remain regarding the cell populations most effective for generating and sustaining the required neutrophil numbers.