Human mast cells (HMCs) are derived from a CD34+ pluripotent progenitor cell that is Kit (CD117+), CD13+, FcεRI- and lacks lineage-specific surface markers. Bone marrow and peripheral blood are two tissue sources available for obtaining CD34+ progenitor cells from which to culture HMCs. CD34+ cells can be isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34+ cells may be immediately cultured in serum-free culture media containing recombinant human stem cell factor (rhSCF), rhIL-6, and rhIL-3 (first week only). Weekly hemidepletions and the removal of adherent cells and/or debris enables the investigator to obtain HMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8–10 wk.