Regulatory T cells (Tregs) provide an essential tolerance mechanism to suppress the immune response. Induced Tregs hold the potential to treat autoimmune diseases in adoptive therapy and can be produced with stimulating signals to CD3 and CD28 in presence of the cytokine TGF-β and IL-2. This report examines the modulation of human Treg induction by leveraging the ability of T cells to sense the mechanical stiffness of an activating substrate. Treg induction on polyacrylamide gels (PA-gels) was sensitive to the substrate's elastic modulus, increasing with greater material stiffness. Single-cell RNA-Seq analysis revealed that Treg induction on stiffer substrates involved greater use of oxidative phosphorylation (OXPHOS). Inhibition of ATP synthase significantly reduced the rate of Treg induction and abrogated the difference among gels while activation of AMPK (AMP-activated protein kinase) increased Treg induction on the softer sample but not on the harder sample. Treg induction is thus mechanosensitive and OXPHOS-dependent, providing new strategies for improving the production of these cells for cellular immunotherapy. Keywords: Mechanosensing; Metabolism; Transcriptomes; Treg induction.