We have developed a new passaging technique for the expansion of human embryonic stem cells (hESCs) that involves simply pipetting portions of hESCs acquired from colonies, reducing the laborious and time-consuming steps in the expansion of hESCs. Compared to general mechanical methods of passaging, our pipetting method allowed hESCs colonies to be broken into small fragments, which showed significantly higher attachment rates onto feeder cell layers. This technique produced three times the number of hESCs colonies than conventional mechanical methods. In addition, this pipetting method allowed us to distinguish differentiated hESCs from undifferentiated hESCs during hESCs colony pipetting. The hESCs cultured by pipetting method displayed normal human chromosomes for over 60 passages. According to RT-PCR and immunohistochemical analysis, the hESCs successfully maintained their undifferentiated state and pluripotency which was also confirmed by teratoma formation in viva Therefore, the pipetting method described in this study is a useful tool to efficiently and quickly expand hESCs on a large scale without enzyme treatment.