ID-8 reversibly maintains the long-term culture of ESCs by sustaining Nanog gene expression through Sox2-Oct3/4 activation. ID-8 enhances Wnt-mediated hESC survival and proliferation via inhibition of DYRKs. The mechanism study indicates that ID-8 maintains the undifferentiated state in the presence of Wnt by modulating Wnt/β-catenin signaling and enhancing the CBP/β-catenin association in hESCs.