MafB is a conserved transcriptional regulator of macrophage development and functional identity across tissues and species.

Homeostatic resident tissue macrophages (RTMs) not only exhibit diversity across tissues but also share common features that are driven by conserved transcriptional programs. While the transcription factor MafB is highly expressed in macrophages, its role in establishing RTM identity and functions remains unclear. Here, we show that MafB was required for the development of bone-marrow-derived macrophages (BMDMs) and most RTMs in vivo. MafB deficiency retained RTMs in a CD52(high) immature stage and disrupted global and tissue-specific identities and functions, impairing phagocytosis, splenic iron recycling, and lung, kidney, and gut physiology. Epigenetic profiling revealed that MafB directly regulated key RTM genes in mice and humans, including Csf1r, Mertk, Fcgr1, Cd163, and Zeb2. In silico analyses further demonstrated strong evolutionary conservation of MafB binding sites across vertebrates. Together, these findings establish MafB as a crucial regulator of RTM development and functional identity, linking MafB-dependent transcriptional programs with defining features of RTMs and tissue homeostasis.