Endothelial cells lining the vasculature are constantly exposed to mechanical forces, including fluid shear and tension at the sites of adhesion to neighbouring cells and the extracellular matrix (ECM). Signals transmitted by mechanosensory complexes cause rapid changes in the structure and dynamics of the cytoskeleton and in turn, drive cell polarity, shape, migration and differentiation. However, how different mechanical stimuli are integrated at a single cell level remains poorly understood. Previous work from our group revealed that the atypical cadherin FAT4 regulates lymphatic endothelial cell (LEC) polarity, which is important for vessel elongation and valve morphogenesis. FAT4 is re-distributed in LECs upon exposure to laminar flow and is required for flow-induced changes in cell shape. This discovery explained why mutations in FAT4 cause a human primary lymphoedema syndrome characterised by lymphatic dysplasia and lymphoedema. Here, we show that lack of response to flow in FAT4-deficient LECs is underpinned by impaired remodelling of the cytoskeleton. FAT4-deficient LEC monolayers exposed to laminar flow in vitro display less aligned, more disorganised actin fibres and microtubules. Imaging of the lymphatic vasculature in vessels in FAT4-deficient mouse embryos revealed redistribution of F-actin to the cell periphery and more disorganised microtubule networks, compared to littermate controls. Loss of FAT4 leads to changes in the subcellular localisation of RhoA GTPase’s activity and the actin depolymerising factor cofilin, both in the presence and absence of flow. Collectively, our data indicate that FAT4 relays mechanical signals in endothelial cells by orchestrating cytoskeleton remodelling in response to shear stress and tension. Current work aims to further define the molecular mechanisms underlying FAT4-mediated control of the cytoskeleton.