Aims Bone morphogenetic protein (BMP) signalling plays a key role in regulating the development of the atrioventricular (AV) septum and valves; however, the molecules that mediate the complex activities of BMP signalling are not fully understood. The major goal of this study is to identify the critical downstream regulatory targets of BMP signalling in AV cushions, which are precursors of the AV septum and valves. Methods and results We established a conditional immortal AV cushion mesenchymal cell line, tsA58-AVM. Using this line, we observed that the expression of Sema6D is upregulated by BMP stimulation through microarray analysis. Sema6D is required for BMP-upregulated migration of tsA58-AVM cells. To reveal the in vivo role of Sema6D, we established the Sema6Dloxp/loxp mouse line and specifically inactivated Sema6D in endocardial cells (by Nfatc1Cre) when cushion mesenchymal cells started to form. We observed a hypocellular AV cushion defect in mutant hearts at early stages (E9.25, E9.5). The defect was resolved at a later stage, most likely due to compensation by increased Sema6C in mutant AV cushions. Furthermore, our ex vivo culturing and in vivo transgenic studies collectively suggest that SEMA6D activates Rho through PLXNA1-FARP1 to promote cushion mesenchymal cell formation. Conclusions We demonstrate for the first time that Sema6D is a target of BMP signalling and that Semaphorin signalling is essential for the initiation of cushion mesenchymal cell formation in the AV canal. Our study reveals a novel BMP-Sema6D-Rho axis regulating AV cushion development.