During embryogenesis,hematopoietic stem progenitor cells (HSPCs) are believed to be derived from hemogenic endothelial cells (HECs).Moreover,arterial feature is proposed to be a prerequisite for HECs to generate HSPCs with lymphoid potential.Although the molecular basis of hematopoietic stem cell-competent HECs has been delicately elucidated within the embryo proper,the functional and molecular characteristics of HECs in the extraembryonic yolk sac (YS) remain largely unresolved.In this study,we initially identified six molecularly different endothelial populations in the midgestational YS through integrated analysis of several single-cell RNA sequencing (scRNA-seq) datasets and validated the arterial vasculature distribution of Gja5+ECs using a Gja5-EGFP reporter mouse model.Further,we explored the hemogenic potential of different EC populations based on their Gja5-EGFP and CD44 expression levels.The hemogenic potential was ubiquitously detected in spatiotemporally different vascular beds on embryonic days (E)8.5-E9.5 and gradually concentrated in CD44-positive ECs from El0.0.Un-expectedly,B-lymphoid potential was detected in the YS ECs as early as E8.5 regardless of their arterial features.Furthermore,the capacity for generating hematopoietic progenitors with in vivo lymphoid potential was found in nonarterial as well as arterial YS ECs on E10.0-E10.5.Importantly,the distinct identities of E10.0-E10.5 HECs between YS and intraembryonic caudal region were revealed by further scRNA-seq analysis.Cumulatively,these findings extend our knowledge regarding the he-mogenic potential of ECs from anatomically and molecularly different vascular beds,providing a theoretical basis for better understanding the sources of HSPCs during mammalian development.