AbstractMesenchymal stem cells (MSCs) closely interact with the immune system, and they are known to secrete inflammatory cytokines in response to stress stimuli. The biological function of MSC-derived inflammatory cytokines remains elusive. Here, we reveal that even under physiological conditions, MSCs produce and release a low level of tumor necrosis factor alpha (TNFα), which is unexpectedly required for preserving the self-renewal and differentiation of MSCs via autocrine/paracrine signaling. Furthermore, TNFα critically maintains MSC function in vivo during bone homeostasis. Mechanistically, we unexpectedly discovered that physiological levels of TNFα safeguard MSC homeostasis in a receptor-independent manner through mechanical force-driven endocytosis and that endocytosed TNFα binds to mammalian target of rapamycin (mTOR) complex 2 and restricts mTOR signaling. Importantly, inhibition of mTOR signaling by rapamycin serves as an effective osteoanabolic therapeutic strategy to protect against TNFα deficiency and mechanical unloading. Collectively, these findings unravel the physiological framework of the dynamic TNFα shuttle-based mTOR equilibrium that governs MSC and bone homeostasis.