The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34(+) hematopoietic stem cells which require less culturing time than previously reported methods. CD34(+) cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MCTC type or connective tissue-type HMC. Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.