Vitronectin establishes a differentiation-restrictive extracellular microenvironment that sustains myoblast...

The extracellular matrix (ECM) critically regulates cell fate decisions, yet matrix components that actively impose differentiation-restrictive states remain insufficiently defined. Here, we identify vitronectin (VN), a major serum-derived ECM glycoprotein, as a differentiation-restrictive matrix component that stabilizes a proliferation-competent microenvironment for myoblast expansion. Substrate-associated VN suppresses myogenic differentiation while sustaining proliferative activity in an integrin αvβ3-dependent manner. These effects are conserved across multiple myoblast systems, including mouse C2C12, rat L6, human LHCN-M2, and primary embryonic chicken myogenic cells, demonstrating cross-species robustness. Furthermore, we show that serum-dependent differences in VN abundance directly influence myogenic differentiation, indicating that ECM composition is a critical determinant of differentiation outcomes. Under defined serum-free conditions, VN in combination with leukemia inhibitory factor (LIF) supports sustained proliferation while preserving differentiation competence, establishing a controllable expansion platform. Together, these findings redefine vitronectin as an ECM component that enforces a differentiation-restrictive microenvironment and provide a framework for matrix-driven control of progenitor cell fate.