Engraftment of human CD34⁺ hematopoietic stem/progenitor cells into immunodeficient mice leads to robust reconstitution of human T and B cells but not monocytes and macrophages. To identify the cause underlying the poor monocyte and macrophage reconstitution, we analyzed human myeloid cell development in humanized mice and found that it was blocked at the promonocyte stage in the bone marrow. Expression of human M-CSF or GM-CSF by hydrodynamic injection of cytokine-encoding plasmid completely abolished the accumulation of promonocytes in the bone marrow. M-CSF promoted the development of mature monocytes and tissue-resident macrophages whereas GM-CSF did not. Moreover, correlating with an increased human macrophages at the sites of infection, M-CSF-treated humanized mice exhibited an enhanced protection against influenza virus and Mycobacterium infection. Our study identifies the precise stage at which human monocyte/macrophage development is blocked in humanized mice and reveals overlapping and distinct functions of M-CSF and GM-CSF in human monocyte and macrophage development. The improved reconstitution and functionality of monocytes/macrophages in the humanized mice following M-CSF expression provide a superior in vivo system to investigate the role of macrophages in physiological and pathological processes.