Human embryonic stem cells (hESCs) have unique characteristics, i.e., self-renewal and pluripotency, that are clearly distinct from those of other types of cells. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we show that the yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs reacted highly sensitively to YAP downregulation for cell survival. Interestingly, hESC displayed dynamic changes of YAP expression in response to YAP downregulation. Our results also demonstrated that YAP is critical for the maintenance of self-renewal. Meanwhile, we found that doxycycline upregulated YAP in hESCs and attenuated the decrease in cell survival induced by YAP downregulation. However, neither the decrease in the expression of self-renewal markers triggered by YAP downregulation nor neural/cardiac differentiation was affected by doxycycline treatment. Collectively, the results of this study revealed the mechanism underlying the role of YAP as well as a novel function of doxycycline in hESCs.