Extended Data Fig. 1: FACS sorting profiles used in this study.

a, Gating strategy for endothelial and hemogenic endothelial sorts from yolk sac (YS). To determine which populations in the YS contain hemogenic potential, the following populations were sorted and analyzed for hematopoietic activity in vitro: CD41^negCD45^negTER119^negCD31^posKIT^negRunx1b:RFP^pos CD41^negCD45^negTER119^negCD31^posKIT^negRunx1b:RFP^posGfi1:GFP^pos CD41^negCD45^negTER119^negCD31^posKIT^posRunx1b:RFP^pos CD41^negCD45^negTER119^negCD31^posKIT^posRunx1b:RFP^posGfi1:GFP^pos Non-hemogenic endothelial cells (FACS-ENDO) for scRNA-seq were sorted as: CD41^negCD45^negTER119^negCD31^posKIT^negRunx1b:RFP^neg Hemogenic endothelium enriched (FACS-HE) populations for scRNA-seq were sorted as: CD41^negCD45^negTER119^negCD31^posKIT^posRunx1b:RFP^posGfi1:GFP^pos CD41^negCD45^negTER119^negCD31^posKIT^posGfi1:GFP^pos CD41^negCD45^negTER119^negCD31^posKIT^posRunx1b:RFP^posGfi1:GFP^neg b, Gating strategy for erythromyeloid progenitors (EMP) and lymphomyeloid progenitors (LMP) sorts from YS. FACS-EMP: CD3^negB220^negGR1^negTER119^negKIT^posCD127^negCD41^posCD16/32^pos. Bertrand, J.Y. et al. Blood 106, 3004-3011 (2005). Frame, J.M., Fegan, K.H., Conway, S.J., McGrath, K.E. & Palis, J. Stem Cells 34, 431-444, 2016. FACS-LMP: CD3^negB220^negGr1^negTER119^negKIT^posCD127^pos Boiers, C. et al. Cell Stem Cell 13, 535-548, 2013. Yoshimoto, M. et al. Proc Natl Acad Sci U S A 108, 1468-1473, 2011. c, Gating strategy to enrich for HE with LMP potential and HE with EMP potential from YS. FACS-HE^LMP:CD41^negCD45^negTER119^neg CD31^posKIT^posCD24a^posLYVE1^negMCAM^neg FACS-HE^EMP:CD41^negCD45^negTER119^neg CD31^posKIT^posCD24a^negLYVE1^posMCAM^neg d, Gating strategy to analyze phenotypic long-term HSC (LT-HSC) and emerging hematopoietic cells (CD31^posKIT^posCD45^pos) in E11.5 AGM regions and long-term HSC (LT-HSC) in E16.6 fetal livers. LT-HSC (AGM): CD31^posSCA^posKIT^posCD45^posEPCR^pos. LT-HSC (fetal liver): B220^negCD3^negGR1^negTER119^negCD48^negSCA^posKIT^posCD150^pos.