IMPORTANCE Notch proteins are cell surface transmembrane spanning receptors which mediate critically important cellular functions through direct cell-cell contact. Interactions between Notch receptors and their ligands regulate cell fate decisions such differentiation, proliferation and apoptosis in numerous tissues. We have previously shown using immunohistochemistry that Notch1 is localized primarily to the prechondroblastic (chondroprogenitor) layer of the mandibular condylar cartilage (MCC). OBJECTIVE To test if Notch signalling changes patterns of proliferation and differentiation in the MCC and to investigate if Notch signalling acts downstream of Fibroblast Growth Factor 2 (FGF-2). METHODS Condylar cartilage explants were cultured over serum-free DMEM containing either 0 or 50nM DAPT, a Notch signal inhibitor. Explants were used for RNA extraction and immunohistochemistry. RESULTS Analysis of gene array data demonstrated that the perichondrial layer of the MCC is rich in Notch receptors (Notch 3 and 4) and Notch ligands (Jagged and Delta) as well as various downstream facilitators of Notch signalling. Disruption of Notch signalling in MCC explants decreased proliferation (Cyclin B1 expression) and increased chondrocyte differentiation (Sox9 expression). Moreover, we found that the actions of FGF-2 in MCC are mediated in part by Notch signalling. CONCLUSION These data suggest that Notch signalling contributes to the regulation of proliferation and differentiation in the MCC.