Mast cells play a distinct role in the innate immune response. Engineered microenvironments for the express purpose of influencing mast cell activity will provide a novel means of designing biomaterials, as well as a means to systematically investigate mast cell biology in a 3D setting. Here, the effect of nanoscaffolds composed of self-assembling peptides, namely (RADA)4, on bone-marrow-derived murine mast cell (BMMC) activity is reported. Unlike most studies that stimulate mast cells to induce adhesion, this results show that BMMCs spontaneously adhere to the artificial nanoscaffold without initiating their activation. It is observed that the classical immunoglobulin (IgE) antigen-mediated degranulation of adhered BMMC is inhibited by the nanoscaffold, while non-IgE (A23187)-induced degranulation is unaffected. The inhibition of IgE–antigen-mediated degranulation is likely a result of inhibited molecular diffusion within the matrix; antigen diffusion, IgE–FcεRI complex shuttling, and/or formation of multiple IgE–FcεRI clusters may be physically hindered in the presence of the polyvalent nanofiber network. Moreover, the IgE/antigen-induced inflammatory cytokine tumor necrosis factor α release from adherent BMMCs is significantly reduced likely due to interaction with the nanofiber matrix. This work is considered the first step in quantifying mast cell activity in artificial matrices composed of self-assembling peptides.