One of the mechanisms by which tumor cells evade the immune system is the lack of proper antigen-presenting cells. Improvement in host immunity against tumor cells can be achieved by promoting the differentiation of dendritic cells (DCs) from immature myeloid cells (Gr-1(+)Ly-6C(+)F4/80(+)) that accumulate in the bone marrow and lymphoid organs of mice with large tumor burdens. The enriched immature myeloid cells inhibit T-cell proliferation and tumor-specific T-cell response, which can be reversed by the differentiation of immature myeloid cells or depletion of F4/80(+) cells. Sorted Gr-1(+)/F4/80(+) immature myeloid cells differentiated into CD11c(+) cells that express CD80 and I-A/I-E (MHC class II) in the presence of recombinant murine granulocyte macrophage colony-stimulating factor (GM-CSF). Furthermore, intratumoral gene delivery of GM-CSF not only promoted the differentiation of carboxyfluoroscein succinimidyl ester-labeled immature myeloid cells into CD11c(+) cells with the characteristics of mature DCs (CD80(+), I-A/I-E(+)) but also enhanced innate natural killer and adaptive cytolytic T-cell activities in mice treated with interleukin (IL)-12 and anti-4-1BB combination therapy. More importantly, intratumoral delivery of GM-CSF and IL-12 genes in combination with 4-1BB costimulation greatly improved the long-term survival rate of mice bearing large tumors and eradicated the untreated existing hepatic tumor. The results suggest that inducing the maturation of immature myeloid cells, thus preventing their inhibitory activity and enhancing their antigen-presenting capability, by GM-CSF gene therapy is a critically important step in the development of effective antitumor responses in hosts with advanced tumors.