We attempted the phenotypic characterization of peripheral blood (PB) cells after daily administration of macrophage colony-stimulating factor (M-CSF) in mice. The number of CD11b+ cells was increased by M-CSF treatment (2- and 5-day injections). Notably, CD11bbrightCD11cdim, CD11b+CD11c+ and CD11b+CD80+ cells were significantly increased by 2-day treatment of M-CSF. On the other hand, the number of NK1.1+ cells was not changed by the 2-day treatment, but it was significantly increased by the 5-day treatment. However, the numbers of CD3+ and NK1.1+CD3+ cells were not changed by M-CSF treatment. Then, mononuclear cells (MNCs) were separated from the PB of mice treated with saline or M-CSF, and they were incubated with GM-CSF + IL-4 or IL-2. Compared with the saline-treated one (S-MNCs), the MNCs of M-CSF-treated mice (M-MNCs) showed strong proliferation by the GM-CSF + IL-4 stimulation. The MNCs could stimulate proliferation of allo-T cells in the mixed lymphocyte reaction (MLR), especially the M-MNCs showed strong reaction. On the other hand, the stimulation by IL-2 induced strong cell growth of MNCs. And M-CSF treatment enhanced this response. Furthermore, the M-MNCs (stimulated by IL-2 in vitro) exhibited greater cytotoxicity against Yac-1 cells than the S-MNCs. In conclusion, we found that administration of M-CSF mobilized CD11b+, CD11b+CD11c+, CD11b+CD80+, and NK1.1+cells into PB. And the injection of M-CSF facilitates the generation of dendritic and natural killer cells from PB cells in vitro. These results suggest that the mobilized cells may provide for application of immunotherapy.