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.