Significance The process of trabeculation is central to heart development and maturation, as it allows the increase in muscle mass before the formation of coronaries. This complex process involves a number of morphological changes in a subset of cardiomyocytes, resulting in their delamination from the compact layer. As cardiomyocytes delaminate, they must also remain attached to the compact layer. We identified Erb-b2 receptor tyrosine kinase 2-mediated relocalization of N-cadherin (Cdh2) as a mechanism underlying the formation of cell–cell junctions between trabecular and compact layer cardiomyocytes. Interestingly, we found that blood flow and heart contraction are also essential for the localization of Cdh2 molecules. These studies further our understanding of the complex cell biological processes underlying the maturation of the vertebrate heart.