The health risks of a dysregulated immune response during spaceflight are important to understand as plans emerge for humans to embark on long-term space travel to Mars. In this first-of-its-kind study, we used adoptive transfer of T-cell receptor transgenic OT-II CD4 T cells to track an in vivo antigen-specific immune response that was induced during the course of spaceflight. Experimental mice destined for spaceflight and mice that remained on the ground received transferred OT-II cells and cognate peptide stimulation with ovalbumin (OVA) 323-339 plus the inflammatory adjuvant, monophosphoryl lipid A. Control mice in both flight and ground cohorts received monophosphoryl lipid A alone without additional OVA stimulation. Numbers of OT-II cells in flight mice treated with OVA were significantly increased by 2-fold compared with ground mice treated with OVA, suggesting that tolerance induction was impaired by spaceflight. Production of proinflammatory cytokines were significantly increased in flight compared with ground mice, including a 5-fold increase in IFN-gamma and a 10-fold increase in IL-17. This study is the first to show that immune tolerance may be impaired in spaceflight, leading to excessive inflammatory responses.