In addition to their contribution to research fields such as early human development, self-renewal, and differentiation mechanisms, human embryonic stem cells (hESCs) may serve as a tool for drug testing and for the study of cell-based therapies. Traditionally, these cells have been cultured with mouse embryonic fibroblast (MEF) feeder layers, which allow their continuous growth in an undifferentiated state. However, for future clinical applications, hESCs should be cultured under defined conditions, preferably in a xeno-free culture system, where exposure to animal pathogens is prevented. To this end, different culture methods for hESCs, based on serum replacement and free of supportive cell layers, were developed. This chapter discusses a simple, feeder-free culture system on the basis of medium supplemented with transforming growth factor beta1 (TGFbeta1), basic fibroblast growth factor (bFGF) and fibronectin as matrix.