Activation of Notch signaling within developing EBs by Jagged1. (A) The activation of Notch signaling during hematopoietic hEB development was assessed using whole mount immunofluorescent staining of hEBs stimulated with (+) or without (–) Jag1, to detect Jagged1 (upper panels), Notch1 (middle panels), and HES1 (bottom panels). Higher numbers of positive cells for each factor were observed in Jag1-treated hEBs (+, right panels). Scale bars represent 100 μm. (B) Quantification of Jagged1-, Notch1-, or HES1-positive hEBs in the presence or absence of Jag1. **P < .01 (n = 3). (C) Flow cytometric analysis represented increase of Notch1-expressing cells in day 10 EBs upon Jag1 stimulation compared with control. (D) Western blot analysis of Notch-signaling pathway components, including full-length Notch1, NICD, and HES1 in undifferentiated hESCs and developing hematopoietic EB development, in the presence or absence of Jag1. Notch1-positive brain extract was used as a positive control.