Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSCs). In cancer patients, increased MDSCs correlate with more aggressive disease and a poor prognosis. Expression of 15 immune factors (TGFβ, IL-1β, IL-4, IL-6, IL-10, GM-CSF, M-CSF, IDO, fms-related tyrosine kinase 3 ligand, c-kit ligand, inducible NO synthase, arginase-1, TNF-α, cyclo-oxygenase 2, vascular endothelial growth factor [VEGF]) by MDSC-inducing human solid tumor cell lines was evaluated by RT-PCR. Based upon these data, cytokine mixtures were then tested for their ability to generate suppressive CD33+ cells from healthy donor PBMCs in vitro by measuring their ability to inhibit the proliferation of, and IFN-γ production by, fresh autologous human T cells after CD3/CD28 stimulation. Induced MDSCs were characterized with respect to their morphology, surface phenotype, and gene expression profile. MDSC-inducing cancer cell lines demonstrated multiple pathways for MDSC generation, including overexpression of IL-6, IL-1β, cyclo-oxygenase 2, M-CSF, and IDO. CD33+ cells with potent suppressive capacity were best generated in vitro by GM-CSF and IL-6, and secondarily by GM-CSF+ IL-1β, PGE 2, TNF-α, or VEGF. Characterization studies of cytokine-induced suppressive cells revealed CD33+ CD11b+ CD66b+ HLA-DR low IL-13Rα2 int large mononuclear cells with abundant basophilic cytoplasm. Expression of inducible NO synthase, TGFβ, NADPH oxidase, VEGF, and/or arginase-1 was also upregulated, and Transwell studies showed suppression of autologous T cells to be contact dependent. Suppressive CD33+ cells generated from PBMCs by GM-CSF and IL-6 were consistent with human MDSCs. This study suggests that these cytokines are potential therapeutic targets for the inhibition of MDSC induction in cancer patients.