T cell activation is modulated by signaling molecules on the surface of antigen-presenting cells (APC); however, in recent years, it has become increasingly clear that cellular forces have a crucial role in T cell activation and subsequent effector responses. Therefore, understanding mechanical modulators is critical in advancing current immunotherapy approaches. To address underlying questions, we engineered a biomimetic system to recapitulate the immune synapse, which is the interface of APC-T cell interaction, using polyacrylamide hydrogels with a defined stiffness range comparable to APC stiffness. The hydrogels were functionalized with different ratios of immobilized anti-CD3 (aCD3) and anti-CD28 (aCD28) antibodies. Our results showed that T cell proliferation, cytokine secretion, and intracellular signaling were all reduced at lower gel stiffness. We observed similar results in our cells’ models, in which APCs with reduced cell stiffness induced lower T cell activation. To enhance the physiological relevance of the biosystem, we fabricated cell-sized microbeads of varying stiffnesses, then embedded them in 3D collagen matrices. Overall, our biosystem allows decoupling of biophysical and biochemical interactions in T cells activation in a physiologically relevant microenvironment.