INTRODUCTION Embryonic development and tissue homeostasis depend on cooperation between specialized cell types. Resident macrophages are professional phagocytes that survey their surroundings; eliminate unfit cells, microorganisms, and metabolic waste; and produce a large range of bioactive molecules and growth factors. Resident macrophages also serve tissue-specific purposes: For example, microglia in the central nervous system support neuronal circuit development, Kupffer cells scavenge blood particles and dying red blood cells in the liver, and alveolar macrophages uptake surfactant and remove airborne pollutants and microbes from the airways. Resident macrophage diversity in adult mice is reflected in tissue-specific gene expression profiles, which may be due to responses to specific cues from their microenvironment, different developmental processes, and the contribution of distinct progenitors cell types. Altogether, the mechanisms responsible for the generation of tissue-resident macrophage diversity remain unclear. RATIONALE Tissue-resident macrophages originate, at least in part, from mesodermal erythro-myeloid progenitors (EMPs) from the yolk sac, which invade the embryo proper at the onset of organogenesis. These tissue-resident macrophages are also self-maintained in postnatal tissues, independently of definitive hematopoietic stem cells (HSCs) in a steady state. We therefore hypothesized that resident macrophages represent a founding cell type within most organ anlagen. In this model, the generation of macrophage diversity, as observed in the tissues of postnatal mice, may be integral to organogenesis. RESULTS To test this hypothesis and explore the molecular basis of macrophage diversity in mammals, we performed a spatiotemporal analysis of macrophage development in mice, from embryonic day 9 (E9) to 3 weeks after birth. Unbiased single-cell RNA sequencing (RNA-seq) analysis of CD45 ⁺ cells, combined with RNA-seq analyses of sorted cell populations, genetic fate mapping, and in situ analyses, revealed that EMPs give rise to a population of premacrophages (pMacs) that colonize the whole embryo from E9.5, as they acquire a core macrophage differentiation program that includes pattern recognition, scavengers, and cytokine receptors. The chemokine receptor Cx3cr1 is up-regulated in pMacs and is important for embryo colonization, which is delayed in Cx3cr1 -deficient embryos. Fate mapping of pMacs using a Tnfrsf11a –Cre reporter labels homogeneously fetal and adult tissue-resident macrophages but not HSCs and their progeny. Transcriptional regulators that identify postnatal tissue-resident macrophages in the brain, liver, kidney, skin, and lung were specifically up-regulated immediately after colonization. These dynamic changes mark the onset of diversification into adult macrophages. We identified Id3 as a Kupffer cell–specific transcriptional regulator. Deletion of Id3 in pMacs resulted in Kupffer cell deficiency but did not affect development of microglia and kidney macrophages. CONCLUSION Our study shows that EMP-derived precursors colonize embryonic tissues and simultaneously acquire a full core macrophage program. This is followed by their diversification into tissue-specific macrophages during organogenesis, likely via the expression of distinct sets of transcriptional regulators. These results indicate that differentiation of tissue-resident macrophages is an integral part of organogenesis and identify a spatiotemporal molecular road map for the generation of macrophage diversity in vivo. Our findings provide a conceptual framework to analyze and understand the consequence(s) of genetic variation for macrophage contribution to development, homeostasis, and disease pathogenesis in different tissues and will support efforts to differentiate specialized macrophages in vitro.