In native tissues, the interaction between cells and the surrounding extracellular matrix (ECM) is reciprocal, as cells not only receive signals from the ECM but also actively remodel it through secretion of cell-derived ECM. However, very little is known about the reciprocal interaction between cells and their secreted ECM within synthetic biomaterials that mimic the ECM for use in engineering of tissues for regenerative medicine or as tissue models. Here, poly(ethylene glycol) (PEG) hydrogels with fully defined biomaterial properties are used to investigate the emerging role of cell-derived ECM on culture outcomes. It is shown that human mesenchymal stromal cells (MSCs) secrete ECM proteins into the pericellular space early after encapsulation and that, even in the absence of material-presented cell adhesion motifs, cell-derived fibronectin enables cell spreading. Then, it is investigated how different culture conditions influence MSC ECM expression in hydrogels. Most strikingly, it is found by RNA sequencing that the fibroblast growth factor 2 (FGF-2) changes ECM gene expression and, in particular, decreases the expression of structural ECM components including fibrillar collagens. In summary, this work shows that cell-derived ECM is a guiding cue in 3D hydrogels and that FGF-2 is a potentially important ECM regulator within bioengineered cell and tissue systems.