Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II^hi and MHC-II^lo tumor-associated macrophage (TAM) subpopulations that originate from Ly6C^hi monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C^hi monocytic precursors. M-CSFR signaling blockade shifted the MHC-II^lo/MHC-II^hi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C^hi monocytes into MHC-II^hi TAMs. In addition, the genetic and functional signatures of MHC-II^lo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II^lo TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II^hi phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade. Cancer Res; 76(1); 35–42. ©2015 AACR.