AbstractDevelopment of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest‐derived cells. Studies in Drosophila have shown that the Slit‐Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2‐5 is required for early ventral restriction of Slit3 and that the T‐box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit‐Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart. Developmental Dynamics 239:3303–3311, 2010. © 2010 Wiley‐Liss, Inc.