G-quadruplexes (G4s) are an alternative secondary structure in DNA, which can form from guanine-rich sequences. They have been demonstrated to play key roles in cancer chromatin organization and epigenetics to influence transcription, DNA replication, nucleosome positioning and DNA methylation. However, their presence and function in embryonic stem cells (ESC) has not yet been determined. In this study, we assess the presence of DNA G4s in human ESCs cells and to ask whether they have roles in pluripotency and differentiation. Using G4-ChIP-seq, we demonstrate the presence of DNA G4s in developmental genes and regulatory regions in human ESCs. Upon ESC differentiation, changes in the DNA G4 landscape were observed accompanying the progressive loss of cellular plasticity. We therefore propose that DNA G4s are part of the intricate array of cis-regulatory features controlling human stem cell identity and differentiation potential.