We examined the roles of Notch signaling in the chondrogenesis of mouse mesencephalic neural crest cells. The present study demonstrated that the activation of Notch signaling or the treatment with fibroblast growth factors (FGFs) promotes the differentiation of proliferative and prehypertrophic chondrocytes expressing collagen type 11. Notch activation or FGF2 exposure during the first 24 It in culture was critical for the differentiation of proliferative and prehypertrophic chondrocytes. The expression of SOX9, a transcription activator of collagen type 11, was also upregulated by Notch activation or FGF2 treatment. The promotion of proliferative and prehypertrophic chondrocyte differentiation by FGF2 was significantly suppressed by the inhibition of Notch signaling using Notch-1 siRNA. These results suggest that FGFs activate Notch signaling and that this activation promotes the chondrogenic specification of mouse mesencephalic neural crest cells. Furthermore, we investigated the expression patterns of Notch-1, SOX9, and p75, which is a marker of undifferentiated neural crest cells, in the mandibular arch where mesencephalic neural crest cells colonize and undergo chondrogenesis. These in vivo observations, coupled with the results of the present in vitro study, suggest that Notch signaling as well as FGFs is a component of epithelial mesenchymal interactions that promote the chondrogenic specification of mouse mesencephalic neural crest cells. (c) 2006 Elsevier Ireland Ltd. All rights reserved.