Natural killer (NK) cell differentiation from pluripotent CD34+ human hematopoietic stem cells or oligopotent lymphoid progenitors has already been reported. In the present study, long-term cultures of the CD56−/CD34−myeloid-like adherent cell fraction (ACF) from umbilical cord blood (UCB), characterized by the expression of CD14+ as well as other myeloid markers, were set up with flt3 ligand (FL) and interleukin-15 (IL-15). The UCB/ACF gradually expressed the CD56 marker, which reached fairly high levels (approximately 90% of the cells were CD56+) by day 15. FL plus IL-15–driven ACF/CD56+ cells progressively expressed a mature NK functional program lysing both NK- and lymphokine-activate killer (LAK)–sensitive tumor targets and producing high levels of interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and IL-10 upon stimulation with IL-12 and IL-18. Similar results were obtained when highly purified CD14+ cells from UCB were cultured with FL and IL-15. In contrast, UCB/CD34+ cells cultured under the same conditions showed a delayed expression of CD56 and behaved functionally differently in that they exhibited NK but not LAK cytotoxicity and produced significantly fewer cytokines. Kinetic studies on the phenotype of UCB/ACF or UCB/CD14+ cells cultured in the presence of FL and IL-15 showed a rapid decrease in CD14 expression after day 5, which reached levels of zero by day 20. Approximately 60% of the CD56+ derived from the UCB/ACF or the UCB/CD14+ cells coexpressed CD14 by day 5. Taken together, our data support the role of CD14+ myeloid-like cells within UCB as a novel progenitor for lymphoid NK cells.