Transforming Growth Factor-β1 (TGFβ1) is a well-established growth factor that regulates tenocyte differentiation, extracellular matrix production, and cell fate. We previously demonstrated its critical role in the formation of in vitro 3D tendon constructs; however, the downstream signaling mechanisms remain unclear. In this study, we investigated the function of mTORC1 (Mammalian Target of Rapamycin Complex 1) and STAT3 (Signal Transducer and Activator of Transcription 3) in TGFβ1-induced 3D tendon formation using rapamycin (mTORC1 inhibitor) and stattic (STAT3 inhibitor). Inhibiting either pathway impaired TGFβ1-induced thickening of tendon construct, cell proliferation, and collagen fibrillogenesis. Molecular analyses confirmed that mTORC1-STAT3 signaling partially mediates TGFβ1-induced Scx expression and tenocyte elongation in the peripheral layer of 3D tendon constructs. Furthermore, TGFβ1 treatment enhanced mTOR and STAT3 phosphorylation while inhibiting mTORC1 signaling reduced TGFβ1-induced STAT3 phosphorylation. These results support the TGFb1-mTORC1-STAT3 signaling axis in 3D tendon constructs. Overall, these findings identify the mTORC1-STAT3 axis as a key mediator of TGFβ1-driven in vitro tendon formation, suggesting its role in tendon maturation and extracellular matrix organization.