Objectives: Original odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice. Methods: A groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1–14 days postoperative. Results: Odontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP–positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1–2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp–dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3–5 to form reparative dentin. Nestin-GFP–positive cells at the pulp–dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(−)/Nestin(+) cells were intermingled in the newly differentiated OBLCs. Conclusions: The commitment of Nestin-GFP–positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp–dentin border and their differentiation into OBLCs.