The development of synthetic and well-defined extracellular matrices that are free of xenogeneic components and are capable of inducing desired cellular responses holds great potential for use in vitro as 3D cell culture environments and in vivo as scaffolds for tissue regeneration. In this study, the impact of free and partially occupied epoxide groups on the viability, activity, and behavior of rat fibroblasts encapsulated in thermoresponsive hydrogels based on poly-(N-isopropylacrylamide) (pNiPAAm) was investigated. While fibroblasts encapsulated in neat pNiPAAm remained rounded and showed significant toxicity by 5 days, those encapsulated in the epoxide-modified thermogels demonstrated not only high viability but also an ability to proliferate, attach, produce extracellular matrix (ECM) components, and cluster. The results demonstrated that the presence of free epoxide groups led to the local conjugation of available proteins to produce a modified structure in situ, which supported cell viability, activity, and cluster formation within the hydrogel.