Notch1-deficient (Notch1ⁱⁿ³² allele) and γ-secretase inhibitor–treated embryoid bodies (EBs), but not Notch1-deficient embryos (Notch1^Δ1 allele), produce expanded primitive erythroid colony-forming unit (CFU-EryP) progenitors. (A) Kinetic analysis of primitive erythroid progenitor formation from EBs at various stages of differentiation. EBs differentiated in liquid culture for 2.5 to 9 days were assayed for CFU-EryP. Values represent the average of 3 replicate platings. Similar results were obtained in at least 4 independent experiments performed in EBs differentiated for 4 to 6 days. Wild type (+/+), Notch1 heterozygous (+/–), and 2 independently derived Notch1 null (–/–) ES cell lines were examined. (B) Wild-type EBs were treated at 1.5, 2.5, or 3.5 days of differentiation with a single dose of the γ-secretase inhibitor Cpd no. 11 (50 μM) or with the carrier alone, DMSO, as a control. Primitive erythroid progenitors (CFU-EryP) were then assayed at day 5 of differentiation. Values represent the average of 3 replicate platings. Similar results were obtained in an independent experiment. (C) Wild-type EBs were treated at day 3.5 of differentiation with a single dose of the γ-secretase inhibitor, DAPT (1 μM), or with the carrier, DMSO, as a control. Primitive erythroid progenitors (CFU-EryP) were then assayed at day 4.75 of differentiation (P < .01, Student t test). Values represent the average of 3 replicate platings. (D) CFU-EryPs were assayed from Notch1-deficient CD1 embryos (–/–) or heterozygous and wild-type (+/– or +/+) littermate controls from approximately day 7.25 to 8.5 dpc. 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. Similar results not shown in this graph were obtained in additional experiments performed at various embryonic stages. Approx indicates approximately.