Erythro-myeloid progenitors of the yolk sac that originates during early embryo development has been suggested to generate tissue-resident macrophage, mast cell, and even endothelial cell populations from fetal to adult stages. However, the heterogeneity of erythro-myeloid progenitors (EMPs) is not well characterized. Here, we adapt single-cell RNA sequencing to dissect the heterogeneity of EMPs and establish several fate-mapping tools for each EMP subset to trace the contributions of different EMP subsets. We identify two primitive and one definitive EMP subsets from the yolk sac. In addition, we find that primitive EMPs are decoupled from definitive EMPs. Furthermore, we confirm that primitive and definitive EMPs give rise to microglia and other tissue-resident macrophages, respectively. In contrast, only Kit+ Csf1r− primitive EMPs generate endothelial cells transiently during early embryo development. Overall, our results delineate the contribution of yolk sac EMPs more clearly based on the single-cell RNA sequencing (scRNA-seq)-guided fate-mapping toolkit. Display omitted •scRNA-seq profiles of early yolk sac identify primitive and definitive subsets of EMPs•Csf1r− pEMPs generate Csf1r+ pEMPs•Only Csf1r− pEMPs contribute to ECs transiently during early embryogenesis•pEMPs and dEMPs give rise to different tissue-resident macrophages Zhao et al. found that Csf1r− pEMPs can differentiate into Csf1r+ pEMPs, and only Csf1r− pEMPs are responsible for transiently contributing to endothelial cells during early embryogenesis, suggesting that pEMPs and dEMPs give rise to distinct populations of tissue-resident macrophages.