The recent rise of adoptive T cell therapy (ATCT) as a promising cancer immunotherapy has triggered increased interest in therapeutic T cell bioprocessing. T cell activation is a critical processing step and is known to be modulated by physical parameters, such as substrate stiffness. Nevertheless, relatively little is known about how biophysical factors regulate immune cells, such as T cells. Understanding how T cell activation is modulated by physical and biochemical cues may offer novel methods to control cell behavior for therapeutic cell processing. Inspired by T cell mechanosensitivity, we developed a multiwell, reusable, customizable, two-dimensional (2D) polyacrylamide (PA) hydrogel-integrated culture device to study the physicochemical stimulation of Jurkat T cells. Substrate stiffness and ligand density were tuned by concentrations of the hydrogel cross-linker and antibody in the coating solution, respectively. We cultured Jurkat T cells on 2D hydrogels of different stiffnesses that presented surface-immobilized stimulatory antibodies against CD3 and CD28 and demonstrated that Jurkat T cells stimulated by stiff hydrogels (50.6 ± 15.1 kPa) exhibited significantly higher interleukin-2 (IL-2) secretion, but lower proliferation, than those stimulated by softer hydrogels (7.1 ± 0.4 kPa). In addition, we found that increasing anti-CD3 concentration from 10 to 30 μg/mL led to a significant increase in IL-2 secretion from cells stimulated on 7.1 ± 0.4 and 9.3 ± 2.4 kPa gels. Simultaneous tuning of substrate stiffness and stimulatory ligand density showed that the two parameters synergize (two-way ANOVA interaction effect: p < 0.001) to enhance IL-2 secretion. Our results demonstrate the importance of physical parameters in immune cell stimulation and highlight the potential of designing future immunostimulatory biomaterials that are mechanically tailored to balance stimulatory strength and downstream proliferative capacity of therapeutic T cells.