As a first approach to defining the molecular requirements for supporting human hematopoietic stem cell maintenance and differentiation in vitro, we have analyzed and compared the ability of various factors to support the maintenance and initial differentiation of human long-term culture-initiating cells (LTC-ICs), a distinct, rare primitive hematopoietic cell type whose progeny after 5 weeks include cells detectable as colony-forming cells. Normal human marrow cells highly enriched in LTC-ICs (approximately 1% pure) were placed in cultures containing either preestablished, irradiated human marrow adherent feeder layers, or feeders consisting of Steel factor-deficient SI/SI, or normal +/+ murine fibroblasts, or no feeders. In some groups, either Steel factor alone, granulocyte colony-stimulating factor (G- CSF) and interleukin-3 (IL-3), or all three factors combined were also added repeatedly. SI/SI murine fibroblasts were equivalent to +/+ controls and to normal human marrow feeders in supporting both LTC-IC maintenance and clonogenic cell output over a 5-week period. Soluble Steel factor alone could, however, effectively substitute for human marrow feeders to support LTC-IC maintenance, although clonogenic cell output was markedly reduced under these conditions. Conversely, soluble Steel factor with G-CSF and IL-3 or with feeders (or all together) did not further enhance (or depress) LTC-IC maintenance, although under these conditions clonogenic cell output was markedly increased. These findings confirm previous evidence that LTC-IC maintenance and clonogenic cell production are differentially regulated and show for the first time that LTC-IC maintenance can be supported by different nonsynergizing factors that may, but need not, include Steel factor.