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Previous studies showed that cultured mouse trophoblast stem cells (mTSCs) have the most rapid proliferation, normal maintenance of stemness/potency, the least spontaneous differentiation, and the lowest level of stress-activated protein kinase (SAPK) when incubated at 2% O 2 rather than at the traditional 20% O 2 or hypoxic (0.5% and 0% O 2 ) conditions. Switching from 2% O 2 induced fast SAPK responses. Here we tested the dose response of AMP-activated protein kinase (AMPK) in its active form (pAMPK Thr172P) at O 2 levels from 20-0%, and also tested whether pAMPK levels show similar rapid changes when mTSC cultures were switched from the optimal 2% O 2 to other O 2 conditions. There was a delayed increase in pAMPK levels ~6-8 h after switching conditions from 20% to 2%, 0.5%, or 0% O 2 . Altering O 2 conditions from 2% to either 20%, 0.5%, or 0% led to rapid increase in pAMPK levels within 1 h, similar to the previously reported SAPK response in mTSC cells removed from 2% O 2 . Twelve hours of 0.5% O 2 exposure led to cell program changes in terms of potency loss and suppressed biosynthesis, as indicated by levels of phosphorylated inactive acetyl CoA carboxylase (pACC). Phosphorylation of ACC was inhibited by the AMPK inhibitor Compound C. However, unlike other stressors, AMPK does not mediate hypoxia-induced potency loss in mTSCs. These results suggest an important aspect of stem cell biology, which demands rapid stress enzyme activation to cope with sudden changes in external environment, e.g., from least stressful (2% O 2 ) to more stressful conditions.