Adoptive transfer of human hematopoietic stem cells (HSCs) into mice lacking T, B and natural killer (NK) cells leads to development of human-blood lineage cells in the recipient mice (humanized mice). Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, reconstitution of NK cells and myeloid cells is generally poor or undetectable. Here, we show that the poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. When plasmid DNA encoding human IL-15 and Flt-3/Flk-2 ligand were delivered into humanized mice by hydrodynamic tail-vein injection, the expression of the human cytokine lasted for 2 to 3 weeks and elevated levels of NK cells were induced for more than a month. The cytokine-induced NK cells expressed both activation and inhibitory receptors, killed target cells in vitro, and responded robustly to a virus infection in vivo. Similarly, expression of human GM-CSF and IL-4, macrophage colony stimulating factor, or erythropoietin and IL-3 resulted in significantly enhanced reconstitution of dendritic cells, monocytes/macrophages, or erythrocytes, respectively. Thus, human cytokine gene expression by hydrodynamic delivery is a simple and efficient method to improve reconstitution of specific human-blood cell lineages in humanized mice, providing an important tool for studying human immune responses and disease progression in a small animal model.