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The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self‐renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here, we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture‐induced modification was shown to be reversible, as culture in mouse embryonic fibroblast‐conditioned media (MEF‐CM) in subsequent passages allowed mTeSR1‐expanded hESCs to re‐establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF‐CM. Furthermore, the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF‐CM, thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential, which can be modulated by culture conditions in a predictive and quantitative manner. S tem  C ells   2015;33:1142–1152

Reversible Lineage‐Specific Priming of Human Embryonic Stem Cells Can Be Exploited to Optimize the Yield of Differentiated Cells