Both physical and chemical signals in the culture environment mediate stem cell fate. For research and the clinical application of stem cells, the development of defined and xeno-free culture conditions to maintain the undifferentiated growth of human embryonic stem cells (hESCs) is highly desirable. We evaluated the effects of different basal media, glucose concentrations, osmolarity, chemical compounds, and hypoxic conditions on hESC self-renewal using defined and xeno-free culture conditions and molecular characterization of the cultured hESCs. Our findings indicate that a combination of KnockOut™ DMEM basal medium, high glucose concentration, and osmolarity of 320-330 mOsm was optimal for maintaining the self-renewal of hESCs under defined, xeno-free conditions. In addition, specific lipids, retinol, and insulin further enhanced the growth of undifferentiated hESCs. Finally, hypoxic conditions increased proliferation and prevented the spontaneous differentiation of hESCs. Although the transcriptional expression of common genes associated with pluripotency was not affected by hypoxia, the translational expression of Oct4 increased significantly under hypoxic conditions. Our results indicated that hypoxia-induced regulation of cell signaling also involves the activation of the calcium and protein kinase C pathway, and several hypoxia signaling-related genes. We identified important nutrients and growth factors required for a defined, xeno-free culture condition to enable reasonable growth characteristics and to maintain the self-renewal of hESCs. Furthermore, we demonstrated that hypoxia supports hESC self-renewal in culture. The use of defined conditions for culturing hESCs will allow for better understanding of pluripotent stem cell regulation and provide more reproducible results.