Notch1-deficient ES cell lines (Notch1ⁱⁿ³² allele) exhibit no defect in proliferation, embryoid body (EB) plating efficiency or growth, or differentiation of the early flk1⁺ population. (A) ES cell proliferation curve. ES cells were plated on day 0 at a density of 5 × 10⁴ cells/mL in 24-well plates. On subsequent days, samples were trypsinized and total cell numbers counted. Values represent the average of 3 samples and error bars indicate standard deviation (SD). Wild-type (+/+), heterozygous (+/–), and 2 independently derived null ES lines (–/–) are shown. (B-C) ES cells, predifferentiated for 2 days without the STO-neo feeder layer but in the presence of LIF, were trypsinized and plated in methylcellulose without exogenous cytokines (B) or in liquid suspension culture (C), at a density of 2 × 10⁴ cells/mL. Total number of EBs were counted at day 4 (B). At days 6 and 12, EBs were pooled and the maximum diameters of at least 30 EBs were measured for each sample (C). Values represent the average of 3 replicate platings from one experiment and error bars indicate SD. Similar results were obtained for several independent experiments. Wild type (+/+), Notch1 heterozygous (+/–), and 2 independently derived Notch1 null (–/–) ES cell lines. (D) FACS analysis of flk-1 expression in Notch1^–/– and Notch1^+/– EBs differentiated for 3.75 days. EBs were dissociated to single-cell suspension and stained with an antibody to flk-1, conjugated to phycoerythrin. The profile indicated by a dotted line represents unstained EB cells. Similar results were obtained in 2 independent experiments, and with additional Notch1^–/– and control cell lines. Percentage of cells in the M2 window is shown.