In our previous work, we established an in vivo coronal pulp regeneration model in which biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells (BM-MSCs) were implanted in the coronal pulp chamber of pulpotomized rat maxillary first molars. In this study, we investigated the in vivo fate of LacZ-labeled BM-MSCs in our coronal pulp regeneration model. BM-MSCs were nucleofected with pVectOZ-LacZ plasmid encoding β-galactosidase 1 day before implantation, and the LacZ-transfected BM-MSCs were implanted into the pulpotomized pulp chamber with biodegradable preformed scaffold-hydrogel constructs. Empty vector was used as a control. After 3 and 14 days, the molars were retrieved and subjected to β-galactosidase staining. At 3 days, β-galactosidase-expressing cells with a round profile were located mainly around the scaffold. At 14 days, when the pulp-like tissue had been generated, the majority of β-galactosidase-expressing cells were detected under the newly formed dentin bridge-like structure, where nestin-expressing odontoblast-like cells were arranged. Immunoreactivity for dentin sialoprotein, a marker of mature odontoblasts, was strongly detected under the original dentin. No β-galactosidase staining was observed in the control group. Thus, we demonstrated that BM-MSCs survived for 2 weeks after implantation and colonized within the site of potential cytodifferentiation. Our findings indicated that BM-MSCs could differentiate into cells involved in mineralized tissue formation in the functionally relevant region.