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Abstract Recent developments in mesenchymal stromal cell (MSC) therapies have increased the demand for tools to improve their manufacture, including the selection of optimal culture substrate materials. While many clinical manufacturers use planar tissue culture plastic (TCP) surfaces for MSC production, others have begun exploring the use of alternative culture substrates that present a variety of spatial, mechanical, and biochemical cues that influence cell expansion and resulting cell quality. In this review, the effects of culture and material properties distinct from traditional planar TCP surfaces on MSC proliferation, surface marker expression, and commonly used indications for therapeutic potency are examined. The different properties summarized include the use of alternative culture formats such as cellular aggregates or 3D scaffolds, as well as the effects of culture substrate stiffness and presentation of specific adhesive ligands and topographical cues. Specific substrate properties can be related to greater cell expansion and improvement in specific therapeutic functionalities, demonstrating the utility of culture materials in further improving the clinical‐scale manufacture of highly secretory MSC products.