Three‐dimensional (3D) in vitro models enable us to understand cell behavior that is a better reflection of what occurs in vivo than 2D in vitro models. As a result, developing 3D models for extracellular matrix (ECM) has been growing exponentially. Most of the efforts for these 3D models are geared toward understanding cancer cells. An intricate network of cells that engages with cancer cells and can kill them are the immune cells, particularly cytotoxic T lymphocytes (CTLs). However, limited reports are available for 3D ECM mimics to understand CTL dynamics. Currently, we lack ECM mimetic hydrogels for immune cells, with sufficient control over variables, such as stiffness, to fully understand CTL dynamics in vitro. Here, we developed PEG‐based hydrogels as ECM mimics for CTLs. The ECM mimics are targeted to mimic the stiffness of soft tissues where CTLs reside, migrate, and deliver their function. To understand cell‐material interaction, we determined the porosity, biocompatibility, and stiffness of the ECM mimics. The ECM mimics have median pore sizes of 10.7 and 13.3 μm, close to the average nucleus size of CTLs (~8.6 μm), and good biocompatibility to facilitate cell migration. The stiffness of the ECM mimics corresponds to biologically relevant microenvironments such as lungs and kidneys. Using time‐lapse fluorescence microscopy, 3D cell migration was imaged and measured. CTLs migrated faster in softer ECM mimic with larger pores, consistent with previous studies in collagen (the gold standard ECM mimic for CTLs). The work herein demonstrates that the PEG‐based ECM mimic can serve as an in vitro tool to elucidate the cell dynamics of CTLs. Thus, this study opens possibilities to study the mechanics of CTLs in well‐defined ECM mimic conditions in vitro.
