Biomimetic scaffolds have played a major role in the advancements in tissue engineering. In addition to mimicking the stiffness, nanofibrous structure, and biochemistry of the native extracellular matrix (ECM), reproducing the three-dimensional (3D) environment of the ECM has been shown to be extremely important. To this end, we designed a co-assembling peptide-amphiphile hydrogel system containing the fibronectin-mimetic PR_g peptide-amphiphile and an E2 diluent peptide-amphiphile for the entrapment and culture of cells in 3D. The E2 diluent peptide amphiphile was designed to screen charges on the PR_g and increase the kinetics of self-assembly in physiologically relevant solutions. Our study investigated two weight percent formulations, 0.5 and 1.0 wt %, and found that in both, entrapped fibroblasts survived encapsulation, proliferated, and deposited collagen IV and fibronectin ECM proteins. The 0.5 wt % gels had a modulus of 429 Pa and supported significantly more fibroblasts proliferation than the 1.0 wt % gels, which had a modulus of 809 Pa. The 1.0 wt% gels though, supported significantly higher mRNA expression and production of ECM proteins. This result indicates by tuning the wt % of our peptide-amphiphile hydrogels, we can encourage either rapid proliferation or ECM deposition. While peptide-amphiphiles are an attractive material for the design of cell scaffolds due to their ability to self-assemble into nanofibrous hydrogels and incorporate multiple biomimetic peptides, there are some limitations associated with these physical hydrogels. One such limitation is that the kinetics of assembly and the mechanical properties of the resulting hydrogels are dependent upon the peptide sequence. We found that the modulus of multifunctional peptide-amphiphile hydrogels ranged from 1000 to 6500 Pa, which was too broad a range to deconvolute the effect of the peptide signal and the effect of mechanical properties. To simplify our system and study the effects of combining ECM protein-mimetic and growth factor-mimetic peptides on the proliferation and function of pancreatic β-cells, peptide mimetics were covalently immobilized on plates. This study found that β-cells proliferate more and secreted significantly more insulin on peptide-functionalized compared to non-functionalized controls. The specific peptide mimetic or combination of peptide mimetics did not significantly affect insulin secretions, but literature suggests that other signals, including cell-cell signaling may play a more significant role in insulin secretion than ECM protein or growth factor signaling. A poly(ethylene glycol) dimethacrylate (PEGDM) hydrogel was functionalized with the laminin-mimetic IKVAV peptide at a 20 µM concentration to match the modulus of non-functionalized hydrogels. This system was used to determine if peptide-functionalization also had an effect in 3D. We found β-cells in IKVAV-functionalized hydrogels proliferated significantly more than β-cells in non-functionalized scaffolds, but no differences in insulin secretion were observed. Together, these studies demonstrate the ability of peptide mimetics to enhance cell proliferation and function of cells in 2D and 3D culture.