Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass (ICM) of the developing embryo. hESCs culture as cell lines in vitro and possess great potential in such research fields as developmental biology and cell-based therapy, as well as such industrial purposes as drug screening and toxicology. When ESCs were first derived by Thomson and colleagues, traditional methods of immunostaining and culturing, using primary mouse embryonic fibroblasts and medium supplemented by serum were used. Considerable efforts have since led to improved methods for isolating new lines in defined and reproducible conditions. This chapter discusses sources for embryos for ESC isolation, commonly used methods for deriving hESC lines, and a number of possible culture systems.