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Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34⁺ cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c⁺CD16 CD1a/c MDCs from CB CD34⁺ cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33⁺CD14CD15 precursors with a mean of 4 × 10⁶ cells generated from 1–4 × 10⁴ CB CD34⁺ cells or myeloid precursors after 2 weeks. After 8–12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1β, IL-6, tumor necrosis factor (TNF)-α, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34⁺ cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.