Notch1-deficient (Notch1ⁱⁿ³² allele) EBs and early embryos display normal definitive colony-forming unit (CFU) progenitor activity. (A-D) EBs differentiated in liquid culture for 6, 8, or 10 days were trypsinized to obtain single-cell suspensions and replated to assay for the following definitive colony-forming progenitors: (A) burst-forming unit–erythroid (BFU-E), (B) colony-forming unit–macrophage (CFU-Mac) (C) colony-forming unit–granulocyte/monocyte (CFU-GM), or for (D) colony-forming unit–mix (CFU-Mix). Values represent the average of 3 replicate platings and error bars indicate SD. Similar results were obtained in independent experiments from EBs differentiated for 10 or 12 days. Wild type (+/+), Notch1 heterozygous (+/–), and 2 independently derived Notch1 null (–/–) ES cell lines were examined. (E-F) Notch1-deficient embryos or littermate controls were dissected at various stages of development. The entire embryo or the dissected yolk sac was dispersed to a single-cell suspension and assayed in methylcellulose with cytokines for the following definitive colony-forming progenitors: (E) definitive erythroid (BFU-E) and (F) macrophage (CFU-Mac). Each time point represents the average number of CFUs from Notch1-deficient (–/–) or control (+/– or +/+) littermate embryos whose approximate chronologic age was based on morphology and/or somite numbers. Error bars represent SD. Similar results not shown in this graph were obtained in additional experiments performed at various embryonic stages.