Given the significance of hydrogels as cell-instructive materials, it is important to understand how differences in their chemical and physical properties are able to direct cell fate. For example, it remains unclear how different hydrogel cross-linking chemistries and gelation mechanisms influence cell behavior. Here, we report on hyaluronan-tyramine (HA-Tyr) hydrogels prepared either with enzymatic cross-linking using horseradish peroxidase and H2O2 or with visible light (500 nm) triggered gelation. We demonstrate that when hydrogels are polymerized to equivalent Young's moduli, the specific cross-linking chemistry of HA-Tyr hydrogels can have a substantial impact on mesenchymal stem cell (MSC) behavior. MSCs cultured on HA-Tyr hydrogels exhibit increased cell spread areas on enzymatically formed substrates relative to photo-cross-linked matrices. While enzymatically formed hydrogels led to MSCs exhibiting greater cell focal adhesion length, MSCs cultured on the photo-cross-linked matrices exhibited smaller cell spread area and shorter focal adhesion length but generated increased traction stress. These findings highlight the importance of understanding hydrogel cross-linking chemistries when the role of biophysical cues in regulating stem cell fate is investigated.