Tumor-associated macrophages (TAMs) are major cellular components in the tumor microenvironment of oral squamous cell carcinomas (OSCCs). Most of these TAMs derive from circulating monocytes that differentiate in situ. In this work, we show that cell culture media (CM) derived from two OSCC cell lines, H413 and TR146, promote monocyte differentiation into M2 macrophages, characterized by a high expression of CD163, CD206 and a low expression of CD11c, CD86 and HLA-DR. Monocyte-derived macrophages (moMΦ) differentiated by CM from H413 cells (H413-CM) were also unable to activate allogeneic T cells, and inhibited T cell activation and proliferation induced by CD3/CD28 stimulation. By culturing monocytes with fractionated H413-CM, we found that soluble proteins mediated CD163CD206 moMΦ differentiation, discarding a role for small metabolites and extracellular vesicles. Differential proteomic analyses on H413-CM fractions revealed the presence of several proteins, including the complement factor H or plasminogen activator inhibitor 1, as potential candidates to induce CD163CD206 moMΦ differentiation. Finally, RNAseq transcriptomic analyses of H413-CM conditioned moMΦ, identified a expression profile signature involving cytokines and cytokine receptors, which surprisingly included IL2RA (encoding CD25). CD25 enhanced expression was confirmed on H143-CM moMΦ. Collectively, these data indicate that the CM from OSCC cell lines promotes the differentiation of functionally immunosuppressive macrophages resembling TAMs, and contributes to the understanding of how OSCCs create an immunosuppressive cellular environment that favors tumor growth.