Cells interact with the extracellular matrix (ECM) via reciprocal biophysical and biochemical signaling in a process known as mechanotransduction. Mechanotransduction controls multiscale biological processes from cellular proliferation, migration, and differentiation to tissue morphogenesis, pathogenesis, and repair. Cellmatrix interactions are mediated by integrins, focal adhesion (FA) proteins, and the cytoskeleton, which enables cells to sense and transmit mechanical signals from and to their ECM. Vinculin, a FA protein, is considered a primary mediator of mechanotransduction and is responsible for modulating FA assembly and cell-generated traction forces in response to matrix properties. While vinculin has been widely studied, these studies have occurred using systems that poorly recapitulate the native cellular microenvironment and/or lack control over the biophysical and biochemical properties of the ECM, which are critical aspects of cell-matrix interactions. Herein, we describe the development of a new tool for the study of cell-matrix interactions and mechanotransduction. This new platform combines PEG-4MAL synthetic hydrogels and 3D Traction Force Microscopy (3D TFM) to enable the quantitative analysis of 3D vinculin mechanotransduction within a microenvironment that has both physiologically relevant and precisely tunable biophysical and biochemical properties. Using this platform, we have engineered hydrogels with user-defined matrix elasticity and ligand density to study how cells generate stresses and deform their surrounding matrix in 3D with response to varying matrix properties. In addition, variants of the vinculin protein have been used to investigate vinculin’s role in the production of force in 3D. By utilizing a platform that has both physiologically relevant and precisely controlled biophysical and biochemical properties, we have gained powerful and unprecedented insights into the role vinculin plays in 3D cellular and molecular mechanotransduction.