<p>Meckel’s cartilage is a transient yet essential structure in the development of the mammalian mandible, with its midsegment undergoing distinct degradation and potentially contributing to bone formation. However, the mechanisms regulating its fate remain unclear. This study aimed to investigate the spatiotemporal changes and potential osteogenic function of the midsegment of Meckel’s cartilage during mouse mandibular development. Using C57BL/6&#xa0;J mice and a tamoxifen-inducible cartilage-specific lineage tracing model, we conducted morphological staining (Alcian Blue, Safranin O, Masson’s trichrome) and immunofluorescence analyses targeting key transcription factors (Sox9, Runx2, Osterix) and vascular marker CD31 from embryonic day 12.5 to postnatal day 0. The anterior portion of the midsegment displayed endochondral-like ossification, marked by chondrocyte hypertrophy, matrix remodeling, and subsequent osteogenic invasion. A shift from cell proliferation to differentiation was noted as Ki67-positive cells decreased by embryonic day 14.5. Sequential expression of Sox9, Runx2, and Osterix was observed along the chondrocyte maturation axis, with peripheral localization of Osterix-expressing cells in remodeling zones. Endothelial cells expressing CD31 were closely associated with osteoprogenitor cells co-expressing Runx2 or Osterix, particularly on the incisor-facing side, indicating a spatial relationship between vascular structures and osteogenic differentiation. These findings demonstrate that the midsegment of Meckel’s cartilage undergoes an endochondral-like ossification process similar to long bones, involving a Sox9 → Runx2 → Osterix transcriptional cascade and vascular invasion. Thus, our study provides novel evidence of the dynamic ossification of Meckel’s cartilage midsegment, clarifying its role in mandibular development and contributing to the long-standing debate on its evolutionary and developmental significance.</p>