Human ES (hES) cell lines are considered to be a valuable resource for medical research and for applications in cell therapy and drug discovery. For such utilization of hES cells to be realized, however, protocols involved in the use of hES cells, such as those for establishment, propagation, and cryopreservation, have still to be improved. Here, we report on an efficient method for the establishment of hES cell lines and its detailed characterization. Additionally, we developed a new bulk-passaging technique that preserves the karyotypic integrity of hES cell lines when maintained in culture for up to 2 years. Finally, we show that a simplified vitrification cryopreservation technique is vastly superior to standard slow-cooling methods with respect to cell viability. These results provide valuable information that will assist in achieving the goal of the large-scale hES cell culture required for the application of hES cells to disease therapy.