T cell activation is triggered by signal molecules on the surface of antigen‐presenting cells (APC) and subsequent exertion of cellular forces. Deciphering the biomechanical and biochemical signals in this complex process is of interest and will contribute to an improvement in immunotherapy strategies. To address underlying questions, coculture and biomimetic models are established. Mature dendritic cells (mDC) are first treated with cytochalasin B (CytoB), a cytoskeletal disruption agent known to lower apparent cellular stiffness and reduction in T cell proliferation is observed. It is attempted to mimic mDC and T cell interactions using polyacrylamide (PA) gels with defined stiffness corresponding to mDC (0.2–25 kPa). Different ratios of anti‐CD3 (aCD3) and anti‐CD28 (aCD28) antibodies are immobilized onto PA gels. The results show T cell proliferation is triggered by both aCD3 and aCD28 in a stiffness‐dependent manner. Cells cultured on aCD3 immobilized on gels has significantly enhanced proliferation and IL‐2 secretion, compared to aCD28. Furthermore, ZAP70 phosphorylation is enhanced in stiffer substrate a in a aCD3‐dependent manner. The biosystem provides an approach to study the reduction of T cell proliferation observed on CytoB‐treated mDC. Overall, the biosystem allows distinguishing the impact of biophysical and biochemical signals of APC and T cell interactions in vitro.