Prolonged propagation of human embryonic stem (hES) cells is currently achieved by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells. Unlike mouse ES cells, adding growth factors such as leukemia inhibitory factor is insufficient to maintain undifferentiated hES cells without feeder cells. The presence of uncharacterized rodent cells or crude extracts imposes a risk to the clinical applications of hES cells. While others looked for a replacement of MEFs with human fetal cells, we attempted to use easily accessible postnatal human cells such as human marrow stromal cells (hMSCs). Culture-expanded hMSCs from multiple donors were used as feeder cells to support growth of the H1 hES cell line under a serum-free culture condition. Human ES cell colonies cultured on irradiated hMSCs amplified >100-fold during the 30-day continuous culture (in five passages). The longest continuous expansion of hES cells on hMSCs tested to date is 13 passages. The expanded hES cells displayed the unique morphology and molecular markers characteristic of undifferentiated hES cells as observed when they were cultured on MEFs. They expressed the transcription factor Oct-4, a membrane alkaline phosphatase, and the stage-specific embryonic antigen (SSEA)-4, but not the SSEA-1 marker. Expanded hES cells on hMSCs retained unique differentiation potentials in culture and a normal diploid karyotype. The well-studied hMSCs (and this animal cell- and serum-free system) may provide a clinically and ethically feasible method to expand hES cells for novel cell therapies. In addition, this system may help to identify cytokines and adhesion molecules that are required for the self-renewal of hES cells.