We show that loss of β1 integrin function affects connexin (Cx) expression in embryonic stem (ES) cell-derived cardiomyocytes. Both loss of β1 integrin function and inhibition of integrin clustering by RGD peptides in wild type ES cells correlated with upregulated expression of gap junctional proteins in ES-derived cardiomyocytes. The upregulation of connexin transcript levels in β1 integrin-deficient cells is paralleled by a higher fraction of cells co-expressing Cx40 and Cx43. These observations demonstrate that the expression of connexins in developing cardiomyocytes is correlated to integrin-dependent mechanisms. Further, we found that upregulated cardiac connexin expression in β1 integrin-deficient cells is related to Rho- and Wnt-dependent pathways. β1 integrin-deficient cardiac cells displayed high levels of cytoplasmic γ-and β-catenins throughout the differentiation period. The administration of lithium, an activator of β-catenin-dependent pathways, resulted in up-regulated connexin mRNA levels in wild type cardiomyocytes at intermediate, but not at early stages, indicating that the effects are restricted to advanced stages of cardiac differentiation. On the other hand, inhibition of Rho-dependent integrin signaling by treatment of cardiac cells with exoenzyme C3 resulted in immediate up-regulation of cardiac connexin transcript levels at early differentiation stages. Our data indicate that integrin function affects connexin expression in cardiomyocytes via direct effects on RhoA-dependent signaling mechanisms at early differentiation stages and of β-catenin/Wnt-dependent pathways at advanced stages.