Hematopoietic potential is highest in Eng −8/P/lacZ targeted HE cells that do not express surface ENG. (A) Schematic diagram outlining the experimental procedure. FLK1+ mesodermal cells were sorted from day 3 EBs generated from the Eng −8/P/LacZ recombinant ES cell line and cultured in liquid blast culture media. At 48 hours, CD41−/TIE2+/c-KIT+ (HE) cells were sorted into ENG+/LacZ−, ENG+/LacZ+, and ENG−/LacZ+ fractions. These fractions were either directly seeded into CFU-C assays (B) or recultured in LBM for a further 48 hours and analyzed by flow cytometry and CFU-C assays (C). (B) (i-ii) Flow cytometry shows the frequencies of CD41−/TIE2+/c-KIT+ (HE) cells in ENG+/lacZ+, ENG+/lacZ−, and ENG-lacZ+ fractions. (iii) CFU-C potential of each sorted fractions in (i). (C) (i) Flow cytometry analysis of CD41 and TIE2 expression in sorted HE cell fractions after 2 days of reculture in LBM. (ii) Bar chart shows the percentage of CD45− positive cells in sorted fraction. (iii) Bar chart shows hematopoietic colony numbers from each fraction. Primitive and definitive colonies were scored after 4 and 9 days, respectively. Statistical analysis was conducted using Student t test, **P < .01, and ***P < .001.