We used ACCUTASE®, a commercially available cell detachment solution, for single cell propagation of pluripotent hESCs. Unlike trypsin dissociation, ACCUTASE® treatment does not significantly affect the plating efficiency of hESC dissociation into single cells. Cultures dissociated with ACCUTASE® to single cells at each passage maintain a higher proportion of pluripotent cells as compared to collagenase-passaged hESCs. ACCUTASE®-treated hESCs can be grown to a high density as monolayers, and yet retain their pluripotency.