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Bone mesenchymal stem cells (MSCs), as well as cardiomyocytes, are derived from early mesoderm, becoming committed to their fate under the influence of different differentiation factors. We examined whether the overexpression of Tbx18 can induce the differentiation of c-kit+ cMSCs into a phenotype similar to that of native pacemaker cells and whether these transfected cells can couple to adjacent atrial cells with functional consequences. The c-kit+ cMSCs were first sorted, then transfected with different lentiviral vectors. Tbx18-c-kit+ cMSCs represented the experimental group, while EYFP-c-kit+ cMSCs and canine sinoatrial node (SAN) cells were used as controls. Within days of transfection, the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel HCN4 protein and gap junction protein Connexin 45 (Cx45) expression in Tbx18-c-kit+cMSCs were 12-fold and 5.6-fold higher, respectively, than that in EYFP-c-kit+ cMSCs. After co-culture with canine atrial cells in vitro for three days, the funny currents (If) were recorded in the Tbx18-c-kit+ cMSCs, but not in EYFP-c-kit+ cMSCs. The trend of these Ifcurrents was highly similar to that of SAN cells, although the current density was smaller. The Tbx18-EYFP-c-kit+ cMSCs showed responsiveness to β-adrenergic stimulation, and the intracellular cyclic adenosine monophosphate (cAMP) level was higher than that in EYFP-c-kit+ cMSCs. The Tbx18-EYFP-c-kit+ cMSCs delivered fluorescent dye to neighboring atrial cells via gap junctions, thus these cell pairs could communicate as a pacemaker unit. We propose that the overexpression of Tbx18 in c-kit+ cMSCs induces their differentiation to SAN-like pacemaker cells.