Recently emerging evidence has indicated surface nanotopography as an important physical parameter in the stem cell niche for regulating cell fate and behaviors for various types of stem cells. In this study, a substrate featuring arrays of increasing nanopillar diameter was devised to investigate the effects of varying surface nanotopography on the maintenance of undifferentiated human embryonic stem cells (hESC) colonies in the absence of feeder cells. Single hESCs cultured across gradient nanopattern (G-Np) substrate were generally organized into compact colonies, and expressed higher levels of undifferentiated markers compared to those cultured on the unstructured control substrate. In particular, hESC demonstrates a propensity to organize into more compact colonies expressing higher levels of undifferentiated markers towards a smaller nanopillar diameter range (D = 120–170 nm). Cell-nanotopography interactions modulated the formation of focal adhesions and cytoskeleton reorganization to restrict colony spreading, which reinforced E-cadherin mediated cell–cell adhesions in hESC colonies. Maintaining compact hESC colony integrity revealed to be indispensable for hESC undifferentiated state as the loss of cell–cell adhesions within spreading hESC on the control substrate exhibited morphological and gene expression signatures of epithelial-to-mesenchymal transition-like processes. Findings in this study demonstrated a feasible approach to screen the optimal nanotopographical cues for maintaining undifferentiated hESC colonies in feeder free conditions, which provides a platform for further investigations into developing hESC feeder free culture systems for the purpose of regenerative medicine.