Biochemical and mechanical properties of the extracellular matrix (ECM) are known to independently influence cell function. Given the complexity of cellular responses, I hypothesized that the integration of multiple matrix factors as opposed to their individual contribution is key to understanding and controlling cell function. The objective of this thesis was to systematically investigate matrix biochemical and mechanical regulation of multipotent stromal cell (MSC) osteogenesis. First, I demonstrated that substrate stiffness-dependent MSC spreading, proliferation and osteogenic response were differentially regulated by matrix protein type (collagen I vs. fibronectin) and concentration. Second, I developed and characterized a matrix microarray platform that enabled the efficient screening of multiple matrix-derived cues (substrate stiffness, ECM type and density). I implemented the matrix microarray platform together with parametric regression models to elucidate novel matrix interactions in directing mouse MSC osteogenic and adipogenic differentiation. Third, I extended the screening study to examine matrix-dependent human MSC osteogenesis. Non-parametric regression models were used to provide a nuanced description of the multi-factorial matrix regulation in MSC osteogenesis. The response surfaces revealed a biphasic relationship between osteogenesis and substrate stiffness, with the exact location and magnitude of the optimum contingent on matrix composition. Guided by the screening results and perturbation to key cytoskeletal regulators, I identified a novel pathway involving Cdc42 in matrix mechanical and biochemical regulation of MSC osteogenesis. Surprisingly, Cdc42 mediated stiffness-dependent MSC osteogenesis independent of ROCK, suggestive of a contractility-independent mechanism in matrix rigidity signal transduction. In summary, the integration of cell-based arrays and statistical modeling has enabled the systematic investigation of complex cell-matrix interactions. This generalizable approach is readily adaptable to other cellular contexts, complementing hypothesis-driven strategies to facilitate non-intuitive mechanistic discovery. Moreover, the improved understanding of matrix-dependent MSC function also has practical relevance to the development of biomaterials for tissue engineering applications.