Objective: Endothelial cell (EC)-induced mesenchymal cell (MC) differentiation toward a mural cell phenotype requires transforming growth factor beta (TGF-β), cell contact and connexin 43 (Cx43)- or Cx45- heterocellular gap junction intercellular communication (GJIC). However, the identity of the communicated signal, the features of Cx43 required, and the possible regulatory mechanisms have not been elucidated and were investigated herein. Methods & Results: To determine whether channel functionality and the major regulatory domain (the carboxyl terminus, CT) of connexin Cx43 are necessary to support EC-induced differentiation, Cx43 deficient MCs (incapable of undergoing EC-induced mural cell differentiation without re-expression of Cx43 or Cx45) were transduced with wild-type (Cx43wt), channel dead, or truncated (Cx43tr-residues 258-382 deleted) versions of Cx43 and their ability to support EC-induced differentiation was assessed. Our data indicate that both channel functionality and presence of the CT domain are both necessary for EC-induced mural cell differentiation. Moreover, expression of Cx40 did not restore ability of MCs to undergo EC-induced mural cell differentiation, despite supporting GJIC. To determine whether (and which) specific regulatory sites in the carboxyl terminus are necessary for EC-induced mural cell differentiation, constructs of Cx43 with serine to alanine substitutions at the mitogen activated protein kinase (MAPK) or protein kinase C (PKC) target sites were introduced into Cx43 deficient MCs and their ability to undergo EC-induced differentiation was tested. The data indicated that the MAPK targeted serines (S255,279,2982) are necessary, while the PKC targeted serine (S368) is dispensable, for this process. To determine whether calcium ions might be the messengers communicated between ECs and MCs, we investigated whether elevation in EC free intracellular calcium concentration (with ionomycin treatment) can replace Cx43-mediated GJIC, activate TGF-β and induce differentiation. Conclusions: Channel functionality, CT domain and the MAPK target sites in Cx43 are all necessary, and neither alone is sufficient, for Cx43-mediated, EC-induced mural cell differentiation. Unlike Cx43, Cx40 is not capable of supporting EC-induced differentiation, despite supporting GJIC. Calcium is unlikely to be the messenger critical to TGF-β activation during EC-induced differentiation, but similar signaling pathways can be initiated. Taken together, these data support a role for connexins in EC-induced differentiation that is complex and goes beyond that of a simple conduit.