This series of 3 videos demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells (video 1), how to passage them to Matrigel feeder cell-free plates (video 2), and how to maintain hESCs by passaging in Matrigel feeder cell-free conditions (video 3). Numerous prior studies showed that maintenance of viable, undifferentiated hESCs requires culture on inactivated MEF feeder cells. However, for many experiments, a pure population of hESCs free of feeder cell contamination is required. To achieve this goal, we have demonstrated how to passage hESCs from MEF feeder plates to feeder cell-free Matrigel plates and how to maintain and continuously passage hESC colonies in feeder cell-free conditions. By following this protocol, a small amount of MEF feeder cell contamination could still exist at passage one on a Matrigel plate, but this contaminant is usually easy to visualize. At Matrigel passage two and beyond, no MEF contamination is usually found, leaving a pure population of hESCs for experiments. Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state during culture.