Could anyone help me to explain why my mouse embryonic cells will begin die when I add some reagents into the cell medium to differentiate ESs? Mouse ES grow on MEF. Cells will be exposed in MCDB 131 medium supplemented with 1.5 g/l sodium bicarbonate, 1×Glutamax, 10 mM final glucose concentration, 0.5% BSA, 100 ng/ ml GDF8), and 3 mM of CHIR-99021 on day 1, day 2 and days almost the same reagents. I will add some new reagents like 0.25 mM ascorbic acid and 50 ng/ml of FGF7 on day 4 and day 5. Day 6- and 7 need to add new reagents like 0.25 mM SANT-1, 1 mM retinoic acid, 100 nM LDN193189, 1:200 ITS-X , and 200 nM TPB. However, my cell will always die from day 1. Sometimes my all ES cells die after day7. Why, please help me and give me some advice.