Shp-2 modulates secondary EB formation and survival. (A) EBs grown for 7 days in primary differentiation culture were harvested, dissociated, and plated into secondary culture for secondary EBs. Secondary EBs were scored on day 7 of secondary culture. Two independent experiments, cultures plated in duplicate; *P < .0001 comparing Shp-2^R1 to Shp-2^Δ and **P = .0004 comparing Shp-2^R2 to Shp-2^Δ. Error bars represent SEM. (B) ES cells were cultured on gelatinized plates for 96 hours without change or supplementation of media followed by trypsinization, staining with annexin V-FITC, propidium iodide (PI), and FACS analysis. Graphic representation of 4 independent experiments. Values for percent annexin V⁺ cells were calculated by adding the values of the upper right quadrant (annexin V⁺/PI⁺) and lower right quadrant (annexin V⁺/PI^–) of the FACS dot plots; *P = .03 comparing WT to Shp-2^Δ cells, and **P = .07 comparing Shp-2^R1 to Shp-2^Δ cells. (C) FACS analysis dot plots from a representative experiment.