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Mesenchymal stem cell (MSC)-based treatments have shown promise for improving tendon healing and repair.MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes.Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation.Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes.As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds.To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin (two other components of the pathway) using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively.We demonstrated that independently disrupting the cytoskeleton and the Rho/ ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology.Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds.