Human embryonic stem (hES) cells have the potential as starting materials for a wide variety of applications in cell therapy, drug discovery and development. However, the challenge is to produce large numbers of well-characterized hES cells that are pluripotent and of high quality. This is needed to be capable of producing future cell therapies that are safe, effective, and affordable for use in routine clinical practice. A major bottleneck is the present requirement for complex culturing regimes that are very labor intensive and unscalable. hES cells have traditionally been grown on feeder layers made from inactivated mouse or human embryonic fibroblasts, in medium containing serum and other nondefined factors. This makes conditions difficult to reproduce over multiple passages. With a view to simplifying culture conditions we have tested a novel proprietary good manufacturing practice-based system that circumvents the use of feeders completely. The system consists of a matrix and a formulated medium that, in combination, demonstrate a reliable and reproducible way to culture hES cells without the use of feeders. We have been able to grow hES cells (Shef3 and Shef6) for over 20 passages, in this system, without loss of pluripotency, capacity to differentiate, or acquisition of karyotypic abnormalities. Furthermore, we have demonstrated the feasibility of propagating hES cells at clonal dilutions from single cells using this system.