Because differentiation of mesenchymal stem cells (MSCs) is enacted through the integration of soluble signaling factors and physical cues, including substrate architecture and exogenous mechanical stimulation, it is important to understand how micropatterned biomaterials may be optimized to enhance differentiation for the formation of functional soft tissues. In this work, macroscopic strain applied to MSCs in an aligned nanofibrous microenvironment elicited cellular and nuclear deformations that varied depending on scaffold orientation. Reorientation of aligned, oriented MSCs corresponded at the microscopic scale with the affine approximation of their deformation based on macroscopic strains. Moreover, deformations at the subcellular scale corresponded with scaffold orientation, with changes in nuclear shape depending on the direction of substrate alignment. Notably, these deformations induced changes in gene expression that were also dependent on scaffold and cell orientations. These findings demonstrate that directional biases in substrate microstructure convey direction-dependent mechanosensitivity to MSCs and provide an experimental framework in which to explore the mechanistic underpinnings of this response.