Dendritic cells (DCs) are the most potent antigen-presenting cells in terms of initiating primary T-cell-dependent immune responses. We devised a 2-step culture method for obtaining sufficient numbers of functional DCs from umbilical cord blood (CB) CD34+ cells. In the first step, CB CD34+ cells were expanded by stimulation with early-acting cytokines such as stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO) to amplify the hematopoietic progenitor cells. In the second step, granulocyte-macrophage colony-stimulating factor and interleukin 4 were added, and incubation was continued for another 5 days to induce differentiation of the expanded cells into DCs. During the first step of culturing with TPO, SCF, and FL, the total numbers of nucleated cells gradually increased, peaking at 4 weeks (245.3-fold). During the second step, expression of CD1a, CD83, and CD86 increased. Electron microscopic findings showed that these cells had cytosolic expansion to form dendrites and major histocompatibility complex class II compartments, which are characteristic of DCs. Functional analyses revealed that these cells had phagocytic activity and were capable of stimulating allogeneic T-cells in vitro.