The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34+ cells collected from healthy donors. CD34+ cells purified from leukapheresis product were seeded at 1 x 10(4) cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34+ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 +/- 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a +CD14 -HLA-DR+, and CD80+CD86+HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a+HLA-DR+ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.