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Highly enriched, pure populations of pluripotent mouse embryonic stem (mES) cells are a prerequisite to downstream experimental manipulations. However, the existing preplating method does not allow complete removal of co-cultured mouse embryonic fibroblast (MEF) feeder cells. The primary objective of the current investigation was to develop and validate a rapid, single-step separation technique for the complete removal of MEF feeder cells from mES cells. A discontinuous density gradient was prepared using Histopaque 1119 at incremental percentages from the top to bottom of a test tube (20, 40, 60, and 100% in culture medium). A suspension of mES cells and MEF feeder cells was layered on top of the gradient. After centrifugation at 400 × g, ES cells and MEF feeder cells were segregated discretely in separate layers at the 40/20% and 100/60% density interfaces, respectively. The mES cells were enriched to a purity of greater than 99% with a recovery rate of greater than 90%. The separation did not alter the viability or the differentiation potential of mES cells. This study validates a simple technique that enables the preparation of highly enriched mES cells that are essentially free of contaminating MEF feeder cells. The discontinuous density gradient separation method is inexpensive, efficient, rapid, and reproducible. The method can be readily scaled-up to accommodate large batch preparations, enabling a broad range of processing needs. Overall, this simple technique significantly expedites the recovery and enrichment of mES cells from MEFs.