Embryonic stem cells potentially self-renew indefinitely and differentiate into all cell types of the three germ layers. While the mouse embryonic stem cells (mESCs) can be grown undifferentiated in culture in the presence of leukemia inhibitory factor (LIF), human embryonic stem cells (hESCs) require feeder cells such as mouse embryonic fibroblasts (MEFs). Mouse or human LIF does not support the undifferentiated growth of hESCs in vitro. The cultivation of hESCs on MEF feeder cells is time-consuming and requires maintenance of two cell cultures simultaneously. Therefore, much interest has been demonstrated in devising an efficient protocol for growing hESCs without a feeder layer. Some reports suggest that the growth of hESCs in feeder free media is possible with the presence of non-physiological concentrations of growth factors such as bFGF. We hypothesized that because hESCs can be maintained and cultured in the presence of conditioned medium (CM; hESC medium incubated over night with the MEF feeder layer), CM contains factor(s) secreted by the MEFs that are responsible for undifferentiated growth in hESCs. To test this hypothesis, we analyzed the CM to isolate component(s) that support undifferentiated growth of hESCs without feeder cells. Results from these experiments showed salt precipitation of the CM yielded a fraction that supported undifferentiated growth of hESCs. This fraction was then subjected to fractionation by anion exchange column chromatography. This resulted in a subfraction which supported undifferentiated growth of hESCs. Analysis by native and SDS-PAGE showed the presence of a single protein in this subfraction. Studies are in progress to further characterize this protein. These studies may lead to devising defined culture medium for hESCs and should accelerate the use of these cells in tissue engineering applications.