AbstractDuring inflammation, monocytes differentiate within tissues into macrophages (mo‐Mac) or dendritic cells (mo‐DC). Whether these two populations derive from alternative differentiation pathways or represent different stages along a continuum remains unclear. Here, we address this question using temporal single‐cell RNA sequencing in anin vitromodel, allowing the simultaneous differentiation of human mo‐Mac and mo‐DC. We find divergent differentiation paths, with a fate decision occurring within the first 24 h and confirm this resultin vivousing a mouse model of sterile peritonitis. Using a computational approach, we identify candidate transcription factors potentially involved in monocyte fate commitment. We demonstrate that IRF1 is necessary for mo‐Mac differentiation, independently of its role in regulating transcription of interferon‐stimulated genes. In addition, we describe the transcription factors ZNF366 and MAFF as regulators of mo‐DC development. Our results indicate that mo‐Macs and mo‐DCs represent two alternative cell fates requiring distinct transcription factors for their differentiation.