Notch signaling is involved in the hematopoietic development of hFib-hPSCs. (A) Notch ligand immobilization augmented hematopoietic differentiation of hFib-hESCs. EBs treated with Matrigel immobilized with soluble Jagged1 (sJag1) or Delta1 (sDll1) during phase 2 of EB development had increased hematopoietic CD45+ blood development. *P < .05 (n = 3). (B-C) HES1 expression was upregulated by Jag1 and Dll1 phase 2 treatment in developing EBs formed from hESCs (B) and hFib-iPSCs (C) compared with IgG-treated control EBs (CTRL). However, control EBs showed higher HES1 expression than undifferentiated hESCs (B) and hFib-iPSCs (C). **P < .01; *P < .05 (n = 3). (D) Time course expression of Notch1, Notch2, and HES1 during hematopoietic EB development. The expression level of Notch1 and HES1 reached steady state by day 6 and then increased gradually and peaked at day 15. However, only minor changes were observed in Notch2 expression. Two different periods, phase 1 (0-5 days) and phase 2 (6-15 days), were identified based on the expression pattern of Notch signals and previous studies. (E) Notch1-mediated signaling regulated blood development of hESCs. Either the transient (phase 1 [0-5 days] or phase 2 [6-15 days]) or continuous (phases 1 and 2 [0-15 days]) suppression of Notch1 (N1) but not Notch2 (N2) by siRNA treatment decreased blood development relative to control scrambled siRNA treatment. *P < .05 (n = 3). (F-I) Hematopoietic development of EBs is regulated by the status of Notch signaling. Notch-signaling activation by adding Jag1 (F,H) or Dll1 (G,I) to developing EBs results in augmented blood differentiation (F-G). Increased or decreased blood formation coincided with up- or downregulation of HES1 (H-I) by either ligand treatment or Notch1 knockdown (siNotch1). Hematopoiesis and Notch signaling could be recovered in only phase 1 Notch1 knockdown EBs by adding Jag1 (F,H) or Dll1 (G,I), whereas neither the hematopoiesis (F-G) nor the expression of HES1 (H-I) could be recovered after long-term (phase 2 only or phases 1 and 2) suppression of Notch1 (+). **P < .01; *P < .05 (n = 3).