Objective: Mesenchymal stem cells (MSC) differentiation into distinct cell lineages is controlled by their local microenvironment (niche) composed of mainly extracellular matrix (ECM) proteins and associated growth factors. Differentiated cells originating from stem cells (progenitor cells), rather than undifferentiated stem cells, offer a promising approach for effective tissue regeneration. However, niche mechanisms regulating MSC lineage differentiation remain to be deciphered. Previously, we reported that cell-free ECM prepared from bone marrow stromal cells promoted significant MSC proliferation and preserved their stemness. Here we have established unique three-dimensional tissue-specific ECM in vitro, mimicking in vivo stem cell niches, to investigate ECM regulation of MSC differentiation.  Methods: Human bone marrow stromal cells, neonatal skin fibroblasts and adipocytes were cultured on tissue culture plates and treated with ascorbic acid to enhance matrix protein synthesis. Cells were then extracted to develop cell-free Bone Marrow- (BM-ECM), Adipose- (Adipose-ECM) and Skin-derived ECM (Skin-ECM). Human bone marrow-derived MSC were cultured on these cell-free tissue-specific ECM in expansion medium (control), adipogenic or osteogenic induction medium. Cell-lineage-specific transcripts were measured by TaqMan PCR at different time points. Results: Under osteogenic conditions, MSC cultured on BM-ECM expressed much higher levels of alkaline phosphatase, collagen type-1 and bone sialoprotein (osteogenic markers). In addition, MSC cultured on Skin-ECM under osteogenic conditions showed no increase in osteogenic markers expression. MSC cultured on Adipose-ECM in both expansion and adipogenic induction medium expressed significantly higher levels of C/EBPα and PPARγ2 (adipogenic markers). MSC cultured on Skin-ECM in expansion medium expressed considerably higher levels of KRT6A (epithelial differentiation marker). Conclusion: We conclude that ECM derived from specific tissues directs lineage-specific differentiation of MSC into distinct cell phenotypes.  ECM unique protein profiles will be further determined using proteomic analysis.