Actomyosin contractility is a potent suppressor of mesoderm induction by human pluripotent stem cells.

Activation of WNT signaling in human pluripotent stem cells efficiently drives lateral mesoderm specification and subsequent cardiomyocyte differentiation. Stabilization of the WNT effector β-catenin induces mesodermal genes such as TBXT (Brachyury) and triggers an epithelial-mesenchymal transition (EMT). Although mechanical forces are essential for embryonic development, the role of actomyosin contractility during human mesoderm specification remains unclear. We show that increasing contractility through constitutively active Rho kinase or myosin light-chain kinase unexpectedly blocks β-catenin-dependent mesoderm induction and prevents EMT. In contrast, pharmacological or genetic suppression of contractility enhances Brachyury expression and advances EMT onset by 24 h. While β-catenin signaling alone promotes colony-level contractility, we find that contractility must be reduced prior to WNT activation to promote mesoderm specification, indicating a sensitization effect at the pluripotent state. Mechanistically, reduced tension decreases junctional β-catenin and increases nuclear active β-catenin, identifying actomyosin contractility as a key regulator of lineage commitment following WNT pathway activation.