(A) CCS regional specialization is driven by local expression of Tbx5-dependent fast conduction network in the VCS, which overlaps underlying Pan-CCS expression of nodal, slow conduction network. (B) VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into a slow, nodal-like system. (C) The balance of Tbx5, a T-box transcriptional activator, versus Tbx3, a T-box transcriptional repressor, determined the molecular and functional output of VCS myocytes. TBX5 binds and directly activates cis-regulatory elements at fast conduction loci, defining the identity of the adult VCS. In the absence of TBX5 or overexpression of TBX3, the expression of the VCS fast conduction channel genes is suppressed.