Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-IIhi and MHC-IIlo tumor-associated macrophage (TAM) subpopulations that originate from Ly6Chi 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 Ly6Chi monocytic precursors. M-CSFR signaling blockade shifted the MHC-IIlo/MHC-IIhi TAM balance in favor of the latter as observed by the preferential differentiation of Ly6Chi monocytes into MHC-IIhi TAMs. In addition, the genetic and functional signatures of MHC-IIlo TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-IIlo 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-IIhi 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.