Traditional methods of human embryonic (ES) cell maintenance have evolved over the last number of years, culminating in a myriad of published protocols with variable proficiencies with different cell lines. Accordingly, there is a need for standardized and reliable protocols that can be used with a broad range of human ES cell lines. The chapter is divided into two sections: 'routine' and 'extended' protocol sections. In the routine section, protocols such as culturing, freezing and thawing human ES cells on mouse feeders, testing for contamination, and disaggregation in standard (serum replacement containing) human ES cell medium are included. This, in addition to work described in the preceeding chapter, should be suffieient to get a novice laboratory proficient in culturing most of the human ES cell lines available. The extended protocol section includes vitrification, culture on human feeders, feeder-free culture, trypinization, culture on matrigel, and controlled rate freezing, and while not as pervasively used as routine protocols are still useful to the veteran human embryonic stem cell laboratory. © 2007 John Wiley & Sons, Ltd.