JAG1, the gene for the Jagged-1 ligand (Jag1) in the Notch signaling pathway, is variably mutated in Alagille Syndrome (ALGS). ALGS patients have skeletal defects, and additionally JAG1 has been shown to be associated with low bone mass through genome-wide association studies. Plating human osteoblast precursors (human mesenchymal stem cells-hMSCs) on Jag1 is sufficient to induce osteoblast differentiation; however, exposure of mouse MSC (mMSC) to Jag1 actually inhibits osteoblastogenesis. Overexpression of the notch-2 intracellular domain (NICD2) is sufficient to mimic the effect of Jag1 on hMSC osteoblastogenesis, while blocking Notch signaling with a ?-secretase inhibitor or with dominant-negative mastermind inhibits Jag1-induced hMSC osteoblastogenesis. In pursuit of interacting signaling pathways, we discovered that treatment with a protein kinase C d (PKCd) inhibitor abrogates Jag1-induced hMSC osteoblastogenesis. Jag1 results in rapid PKCd nuclear translocation and kinase activation. Furthermore, Jag1 stimulates the physical interaction of PKCd with NICD. Collectively, these results suggest that Jag1 induces hMSC osteoblast differentiation through canonical Notch signaling and requires concomitant PKCd signaling. This research also demonstrates potential deficiencies in using mouse models to study ALGS bone abnormalities.