As a result of improved relevance to in vivo physiology, in vitro studies are increasingly performed in diverse, three-dimensional (3D) biomaterials. However, material cell type pairing effects on cytokine availability remain unclear. We cultured five cell types in agarose, alginate, collagen, Matrigel, or RGD-functionalized polyethylene glycol (PEG) hydrogels. We measured 21 cytokines in the conditioned media, and we identified differences in measured cytokine levels that were cell-type-or material-dependent. We further evaluated our data using principal component analysis. Interestingly, component one identified two classes of biomaterials with characteristic cytokine expression levels. Component two identified cell-type-dependent differences in cytokines related to the wound response. Although elements of soluble cytokine availability are shared despite parameter differences, material and cellular properties variably influenced cytokine levels, underlining the influence of biomaterial cell type pairings on in vitro assay outcomes. Relationships between material properties, cellular responses, and cytokine availability in 3D in vitro models warrant further investigation.