Muse cells are SSEA-3-positive pluripotent-like endogenous stem cells found in various tissues, including peripheral blood and organ connective tissue. Their reserve is considered the hypoxic bone marrow. In mesenchymal stromal cell (MSC) cultures, Muse cells comprise several percent of the population. Clinical trials using intravenous administration of Muse cells without genetic modification or differentiation induction have shown significant therapeutic potential. Since Muse cells are a small fraction of MSCs, developing efficient culture methods to increase their proportion while maintaining their stemness is crucial for enhancing efficiency and reducing costs in clinical research. In this study, we investigated the effects of hypoxia on Muse cell proportions, pluripotency gene expression, and metabolism. Hypoxia increased the Muse cell proportion around twofold, driven by HIF2α rather than HIF1α, and enhanced pluripotency gene expression, potentially via microRNA let-7 upregulation. Hypoxia also shifted metabolism from oxidative phosphorylation to glycolysis, linked to maintaining stem cell properties. These findings suggest that hypoxia represents a cost-effective strategy for expanding Muse cells, offering promising potential for clinical applications.