Recently, several reports of lineage-negative (lin⁻) CD34⁻ cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin⁻CD34⁻CD38⁻ cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin⁻CD34⁻CD38⁻ cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin⁻CD34⁻CD38⁻ cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34⁺ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin⁻CD34⁻CD38⁻ pre-CFC). In contrast to lin⁻CD34⁺CD38⁻cells, this response of lin⁻CD34⁻CD38⁻ cells required exposure to all of the 5 growth factors used. Up to 1.7 × 10⁵ lin⁻CD34⁻ adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β₂M^−/− mice. These studies demonstrate unique properties of a rare subset of lin⁻CD34⁻CD38⁻ cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.