Abstract: Objective To establish a culture system for human embryonic stem cell and label the hES cells with GFP by lentivirus. Methods We typically cultured the human ES cells on the feeders or matrigel. Furthermore, we characterized the undifferentiated hES by their ability to form compact clones positive for stage specific embryonic antigen-3 and surface alkaline phosphatase staining. Results Human ES cells cultured both on feeder cell and matirgel expressed typical surface markers like SSEA-3 and alkaline phosphatase. After transfected by lentivirus and hygromycin selection, green fluorescent protein was strongly expressed in human ES cells. Conclusion we succeed in human ES cell culture with feeder cells and matrigel.And got human ES cells expressing GFP continuously.