Differentiation kinetics and surface phenotypes of clonogenic progenitors of hEB-derived primitive and definitive hematopoieses. Line H1 hEB were differentiated in SF medium and BVF2H for 3-20 days, as described under “Methods.” hEB cells at different time points were evaluated for global gene expressions (A) by DNA microarray (Agilent) analysis (see Table S2 for full list), surface marker expression (E), or recultured at indicated time points in CFC assays (B-D). Replicate CFC assay results represent the average, normalized (per 10⁶ single, viable hEB cells plated), for independent experiments done at least 3 times, with all standard deviations less than 5%-10%). (A) Mesoderm marker Brachyury and hemangioblast-associated (SCL/TAL1, RUNX1, GATA2) genes during days 0-10 of hEB differentiation are selectively shown. (B) The kinetics of formation of BL-CFC, (C) MHE clusters, mixed primitive + definitive erythro-myelopoietic colonies (MIXED) colonies, (D) primitive erythropoietic (PRIM ERYTH), and definitive erythropoietic (DEF ERYTH) CFU are shown during days 3-20 of hEB differentiation. Purple vertical dashed line emphasizes that the initiation and pattern of blast colony formation (starting at day 5 of hEB differentiation) which correlates simultaneously with the onset of ACE/BB9 and CD34 hEB expression and also the emergence of primitive multipotent (MIXED), also primitive erythroid (PRIM ERYTH) CFU (E). In contrast, CD34, CD164, CD31, CD43, and CD41a hEB cell peak cell surface expressions patterns correlated more with the emergence of definitive-type CFU from hEB at later time points (orange dashed line).