The complex spatiotemporal organization of cellular and molecular interactions dictates the physiological function of cells. These behaviors are indications of an integrated response to a three-dimensional cellular environment and anchored in cell adhesion on scaffolds. Here, we are able to control interconnected structural, mechanical, and chemical stimuli by dictating the cellular environment through chemical surface modifications, soft lithography, and mechanical deformation. Control of these variables is obtained through the use of an elastomeric membrane chemically modified for cell adhesion with a pressure-driven cell-stretching device which creates a pattern of forces similar to those encountered in physiological environments. Further, the integration of lithographic methods and chemical patterning allows the introduction of space-and time-dependent parameters by combining mechanical stimulation, biochemical regulation, and scaffolding design. The method is applied to stimulate single cells and cell populations to examine cellular response with spatiotemporal control. This research provides the capacity to probe biological patterns and tissue formation under the influence of mechanical stress.