T cells have the ability to recognize and kill specific target cells, giving therapies based on their potential for treating infection, diabetes, cancer, and other diseases. However, the advancement of T cell-based treatments has been hindered by difficulties in their ex vivo activation and expansion, the number of cells required for sustained in vivo levels, and preferential localization following systemic delivery. Biomaterials may help to overcome many of these challenges by providing a combined means of proliferation, antigen presentation, and cell localization upon delivery. In this work, we studied self-assembling Multidomain Peptides (MDPs) as scaffolds for T cell culture, activation, and expansion. We evaluated the effect of different MDP chemistries on their biocompatibility with T cells and the maintenance of antigen specificity for T cells cultured in the hydrogels. We also examined the potential application of MDPs as scaffolds for T cell activation and expansion and the effect of MDP encapsulation on T cell phenotype. We found high cell viability when T cells were encapsulated in noncationic MDPs, O and D, and superior retention of antigen specificity and tumor-reactivity were preserved in the anionic MDP, D. Maintenance of antigen recognition by T cells in D hydrogels was confirmed by quantifying immune synapses of T Cells engaged with antigen-presenting cancer cells. When 3D cultured in anionic MDP D coloaded with anti-CD3, anti-CD28, IL2, IL7, and IL15, we observed successful T cell proliferation evidenced by upregulation of CD27 and CD107a. This study is the first to investigate the potential of self-assembling peptide-based hydrogels as 3D scaffolds for human T cell applications and demonstrates that MDP hydrogels are a viable platform for enabling T cell in vitro activation, expansion, and maintenance of antigen specificity and therefore a promising tool for future T cell-based therapies.