Human embryonic stem cells (hESCs) exhibit pluripotency and indefinite proliferation and are a potential source of cells for transplantation therapies and drug discovery. These applications will require large amounts of hESCs. However, hESCs are difficult to culture and maintain at larger scales, in part because of their low resistance to dissociation during passaging. To circumvent this, we developed a simple and easy method for establishing hESC sublines tolerant of complete dissociation. These cells exhibit high replating efficiency and also high cloning efficiency, and they maintain their ability to differentiate into the three germ layers. Several sublines have no detectable abnormalities in their karyotypes, and they retained their characteristics under feeder-free culture conditions and after freeze-thawing. Thus, these hESC sublines would be valuable for hESC applications.