Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture while retaining their ability to differentiate to any cell type in the body. Conventionally, hESC are cultured either directly on feeders or on an extracellular matrix supplemented with conditioned medium (CM) from feeders. To minimize the risk of xenozootic infections, several sources of primary human feeders have been identified. However, this does not eliminate the risk of contaminating hESC with infectious agents from the donor human feeders. In this study, we evaluated the use of the CD105+ /CD24 hESC-derived mesenchymal stem cell (MSC) line, HuES9.E1, for its ability to support the growth of undifferentiated hESC in feeder and feeder-free cultures. This line was previously reported to be karyotypically stable and phenotypically displayed MSC-like surface antigens and gene transcription profiles. In addition, like adult MSC, HuES9.E1 can be differentiated to adipocytes, osteocytes, and chondrocytes in vitro. When tested for its ability to support hESC growth, it was found that hESC maintained the undifferentiated morphology for >12 continuous passages in coculture with HuES9.E1 and >8 passages in feeder-free cultures supplemented with CM from HuES9.E1. Furthermore, the hESC cultures continued to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, and retained a normal karyotype. When injected into severe combined immunodeficient (SCID) mice, hESC differentiated to form teratomas comprising of tissues representative of the three embryonic germ layers. Potentially, the ability to derive and use autogeneic feeders may provide a safe and accessible source of feeders for the expansion of hESC required in clinical applications.