The present in vitro study revealed marked differences in immunophenotypic expression and ultrastructure among macrophage colony‐stimulating factor (M‐CSF)–derived, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)–derived, and multi‐CSF–derived macrophages. M‐CSF–derived macrophages were larger and had more markedly differentiated intracellular organelles and more cytoplasmic projections than GM‐CSF–or multi‐CSF–derived macrophages. By the combined method of ultrastructural peroxidase cytochemistry and immunoelectron microscopy, ER‐MP12 was demonstrated mainly on blastic cells; ER‐MP20 on promonocytes, monocytes, and immature macrophages; and F4/80 or BM8 on immature and mature macrophages and monocytes. Macrophage heterogeneity was demonstrated to occur at the stage of macrophage precursor cells, and macrophage differentiation was different between bone marrow hematopoiesis and early fetal hematopoiesis. In vivo, F4/80‐ or BM8‐positive (+) macrophages and ER‐MP12 (+) cells developed in the yolk sac prior to the appearance of ER‐MP20 (+) monocytic cell series. These results imply that CSFs are important factors for the generation of phenotypic heterogeneity of macrophage populations not only in bone marrow but also in fetal hematopoiesis, suggesting that there are different pathways of macrophage differentiation. J. Leukoc. Biol. 55: 642–651; 1994.