Macrophages are key players in inflammation and immune responses due to their phenotypic plasticity. This study examined the effects of pooling donor-derived macrophages on their phenotype and function, focusing on murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (hMDMs). Murine BMDMs were generated using L929-conditioned media and compared across single and pooled donors (two-to-five mice). Similarly, hMDMs cultured with M-CSF from individual donors were compared to pooled cultures. Pooling macrophages did not alter core phenotypic markers (CD11b, F4/80, CD64) or functional outputs such as cytokine secretion and nitric oxide production. In hMDMs, pooling reduced variability and led to slightly elevated or more-uniform marker expression. These findings demonstrate that pooling macrophages minimizes inter-individual variability without compromising cellular stability or function, enhancing reproducibility in immunological research while maintaining the option of single-donor studies for personalized analyses.