Background Binding of Slit ligands to their Robo receptors regulates signaling pathways that are important for heart development. Genetic variants in ROBO1and ROBO4 have been linked to congenital heart defects in humans. These defects are recapitulated in mouse models with ubiquitous deletions of the Slit ligands or Robo receptors and include additional heart defects not currently linked to SLIT or ROBO mutations in humans. Given the broad expression patterns of these genes, the question remains open which tissue-specific ligand-receptor interactions are important for the correct development of different cardiac structures. Methods and Results We used tissue-specific knockout mouse models of Robo1/Robo2, Robo4, Slit2 andSlit3 and scored cardiac developmental defects in perinatal mice. Knockout of Robo2 in either the whole heart, endocardium and its derivatives, or the neural crest in ubiquitous Robo1 knockout background resulted in ventricular septal defects. Neural crest-specific removal of Robo2 in Robo1 knockouts showed fully penetrant bicuspid aortic valves (BAV). Endocardial knock-out of either Slit2or Robo4 caused low penetrant BAV. In contrast, endocardial knockout of Slit3 using a newly generated line resulted in fully penetrant BAV, while removal from smooth muscle cells also resulted in BAV. Caval vein and diaphragm defects observed in ubiquitous Slit3 mutants were recapitulated in the tissue-specific knockouts. Conclusions Our data will help understand defects observed in patients with variants in ROBO1 and ROBO4. The results strongly indicate interaction between endocardial Slit3and neural crest Robo2 in the development of BAV, highlighting the need for further studies of this connection.