Abstract The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34 + hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34 + progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34 + hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7–11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-α. By the two-step culture, total nucleated cells were increased 208±66 (mean±SD, n=13), or 94±29 ( n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34 + cells at the time of starting culture. Out of the total nucleated cells, 23±10.4% of cells in CB cell culture and 25±5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a + cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34 + cells using SCF, GM-CSF and TNF-α. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a + DC generated from CD34 + cells, and the phenotypes and functions of these CD1a + DC could be enhanced efficiently by treatment with a calcium ionophore agent.