Scalable, reproducible, low cost and regulatory-friendly technologies must be developed to enable clinical use of human embryonic stem cell (hESC)-based therapeutics. hESC culture methods use complex, animal-derived products, such as mouse feeder layers, Matrigel™, murine laminin or human-derived biological substances as surfaces to which the hESCs attach. Most of these materials are costly, of limited scalability, have batch to batch variability and are a potential source of adventitious agents. For clinical application of hESC-based therapeutics it is highly desirable to have defined, scalable culture systems for production of cells suitable for clinical use. In this study we describe the development of a fully synthetic, xeno-free surface for the culture of undifferentiated hESC. A peptide sequence derived from the active domain of the vitronectin protein was covalently linked to a synthetic acrylate polymer surface to mimic biological ligands for cell adhesion. Self-renewal and pluripotency of multiple hESC lines (H7, H1, H9, and BG01v) cultured on Synthemax Surface was compared to cells grown on Matrigel control surfaces under various defined media conditions (StemPro®, NutriStem™, mTeSR®1, and XVIVO™ 10). Our results demonstrate efficient adhesion and self-renewal of H7, H1, H9 and BG01v/hOG hESCs on Synthemax Surface for up to 20 serial passages in defined media. Importantly, stable proliferation rate, expression of stem cell specific markers (Oct-4, TRA 1-60, SSEA-4), in vitro and in vivo pluripotency and normal karyotype were retained throughout multiple passages on Synthemax Surface. Further, we demonstrated successful scale-up of Synthemax Surface to large culture vessel formats to accommodate the clinical scale production of therapeutic cells. To our knowledge, Synthemax Surface is the only commercially available, synthetic, non-biological surface that supports the long-term, multi-passage expansion of undifferentiated hESC in chemically-defined, xeno-free media. We believe Synthemax Surface will be applicable for both research purposes and scalable manufacturing of hESC-derived cellular therapeutics. Financial disclosure: This abstract was sponsored by Corning Incorporated and Geron Corporation.