Defined pluripotent stem cell culture media is a valuable tool for tracking and analyzing morphological, cell signaling and gene expression changes in human embryonic stem cells. Cultivation of hESC under xeno-free culture settings provides researchers with a consistent and reproducible environment to test experimental hypotheses and move towards translational and clinical research (Richards et al., Nat Biotechnol 20:933-936, 2002; Richards et al., Stem Cells 21:546-556, 2003). One of the primary concerns of the xenogeneic culture is the transfer of foreign pathogens or antigens that induce disease or immune response by the patient. These undesirable by-products may come from the use of murine-derived feeder cells, xenogeneic matrices, or from animal-derived components found in the cell culture medium or matrix used to isolate or expand the stem cells (Beattie et al., Stem Cells 23:489-495, 2005; Koivisto et al., Reprod Biomed Online 9:330-337, 2004). This chapter describes standardized protocols for obtaining consistent and reproducible results for culturing PSC under xeno-free, feeder-free, or feeder-based systems.