Previous reports have demonstrated the growth of undifferentiated human embryonic stem (HES) cells on mouse embryonic fibroblast (MEF) feeders and on laminin- or Matrigel-coated plastic surfaces supplemented with MEF-conditioned medium. These xenosupport systems run the risk of cross-transfer of animal pathogens from the animal feeder, matrix, or conditioned medium to the HES cells, thus compromising later clinical application. Here we show that human fetal and adult fibroblast feeders support prolonged undifferentiated HES cell growth of existing cell lines and are superior to cell-free matrices (collagen I, human extracellular matrix, Matrigel, and laminin) supplemented with human or MEF feeder-conditioned medium. Additionally, we report the derivation and establishment of a new HES cell line in completely animal-free conditions. Like HES cells cultured on MEF feeders, the HES cells grown on human feeders had normal karyotypes, tested positive for alkaline phosphatase activity, expressed Oct-4 and cell surface markers including SSEA-3, SSEA-4, Tra 1-60, and GCTM-2, formed teratomas in severely combined immunodeficient (SCID) mice, and retained all key morphological characteristics. Human feeder#150;supported HES cells should provide a safer alternative to existing HES cell lines in therapeutic applications.