Human embryonic stem (hES) cells have been traditionally cultured on primary mouse embryonic fibroblasts (PMEFs). However, though STO cells have some advantages over PMEFs and human embryonic fibroblasts (hEFs) as feeder cells, they have never been used as feeder cells to establish hES cell lines. In this study, three hES cell lines (Miz-hES1, Miz-hES2, and Miz-hES3) were established from inner cell masses (ICM), using STO as feeder cells. The three hES cell lines had normal karyotypes and expressed high levels of alkaline phosphatase (AP), cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), and transcription factor Oct-4. After culture on STO cells for 2 yr, hES cells maintained the potential to form derivatives of all three embryonic germ layers. Our results show that STO feeder cells have the potential to support the establishment and maintenance of hES cell lines. In addition, our results suggest that laminin may play an important role in maintaining the undifferentiated proliferation of hES cells.