It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells.