The CD41a-GFP ^dimLineage⁻ population is composed of multipotent hematopoietic progenitors. (A-G) CD43⁺ cells were isolated from hESC differentiation at day 14 and cultured for an additional 5 days on hESC-derived ECs; flow cytometry was performed examining multiple parameters as shown in panels B through G. (E) Erythroid derivatives are indicated by colocalization of CD71 and CD235a. (F) Labeling of the CD41a fraction with CD42a and CD61 indicates megakaryocyte identity. (G) Labeling of the CD15⁺ and CD45^bright populations with CD14 and CD33 indicates myeloid identity. (H) CD43⁺ cells were isolated from VPr-mOrange/CD41a-GFP hESCs at day 14 of differentiation and cultured for an additional 5 days on hESC-derived EC feeders, followed by labeling to distinguish Lineage⁻ and Lineage⁺ populations; expression of CD41a-GFP transgene and CD41a surface antigen is shown for all populations. (I-O) CD41a-GFP ^dimLineage⁻ cells indicated in panel H were collected for Megacult (I) and Methocult (J-O) colony assays. (J) The colony-forming potential of Lineage⁻ cells was compared with Lineage⁺ populations, and the number of CFU-GEMM, CFU-E, and CFU-G/M were scored after 2 weeks. (L-O) The CD41a-GFP ^dimLineage⁻ population was sorted as single cells directly into Methocult medium; after 15 days, clones were isolated and labeled and surface phenotype was assessed by flow cytometry. (J) Error bars represent SD among 3 replicates.