Decellularized extracellular matrices (ECM) from in vitro cell cultures can serve as in vivo-like matrix scaffolds for modulating cell-ECM interactions. Macromolecular crowding (MMC), the supplementation of synthetic or naturally occurring molecules resulting in excluded volume effects (EVE), has been demonstrated to provide valuable options for recapitulating the physiological environment of cells during matrix secretion. Human mesenchymal stem cell (MSC)-derived ECM was produced upon supplementation of standard culture medium with three different macromolecules of various size (10-500 kDa). Matrix secretion, ECM morphology and composition were compared for matrices obtained from crowded and non-crowded MSC cultures. In the context of generating functional stem cell niches, the MSC-derived bone marrow mimetic ECM scaffolds were tested for their supportive effect to maintain and expand human hematopoietic stem and progenitor cells (HSPC) in vitro. MMC in combination with metabolic stimulation of MSC was found to result in tissue-specific, highly organized ECM capable of retaining glycosaminoglycans and growth factors to effectively build in vitro microenvironments that support HSPC expansion.