Human embryonic stem cells (hESCs) proliferate infinitely and are pluripotent. Only a few reports, however, describe specific and efficient methods to induce hESCs to differentiate into mature blood cells. It is important to determine whether and how these cells, once generated, behave similarly with their in vivo-produced counterparts. We developed a method to induce hESCs to differentiate into mature neutrophils. Embryoid bodies were formed with bone morphogenic protein-4, stem cell factor (SCF), Flt-3 ligand (FL), interleukin-6 (IL-6)/IL-6 receptor fusion protein (FP6), and thrombopoietin (TPO). Cells derived from the embryoid bodies were cultured on a layer of irradiated OP9 cells with a combination of SCF, FL, FP6, IL-3, and TPO, which was later changed to granulocyte-colony-stimulating factor. Morphologically mature neutrophils were obtained in approximately 2 weeks with a purity and efficiency sufficient for functional analyses. The population of predominantly mature neutrophils (hESC-Neu's) showed superoxide production, phagocytosis, bactericidal activity, and chemotaxis similar to peripheral blood neutrophils from healthy subjects, although there were differences in the surface antigen expression patterns, such as decreased CD16 expression and aberrant CD64 and CD14 expression in hESC-Neu's. Thus, this is the first description of a detailed functional analysis of mature hESC-derived neutrophils.