There is tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work we leverage a methacrylated hyaluronic acid hydrogel platform to study stromal cell mechanotransduction. Hydrogels are first formed through thiol-Michael addition to model normal soft tissue (e.g., lung) stiffness ( E ~ 1 kPa). Secondary crosslinking via radical photopolymerization of unconsumed methacrylates allows matching of early- ( E ~ 6 kPa) and late-stage fibrotic tissue ( E ~ 50 kPa). Early passage (P1) primary human mesenchymal stromal cells (hMSCs) display increased spreading, myocardin-related transcription factor-A (MRTF-A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF-A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models.