Controlling metastasis is the key to improving outcomes for osteosarcoma patients; yet our knowledge of the mechanisms regulating the metastatic process is incomplete. Clearly Fas and Ezrin are important, but other genes must play a role in promoting tumor spread. Early developmental pathways are often recapitulated in malignant tissues, and these genes are likely to be important in regulating the primitive behaviors of tumor cells, including invasion and metastasis. The Notch pathway is a highly conserved regulatory signaling network involved in many developmental processes and several cancers, at times serving as an oncogene and at others, behaving as a tumor suppressor. In normal limb development, Notch signaling maintains the apical ectodermal ridge in the developing limb bud and regulated size of bone and muscles. Here, we examine the role of Notch signaling in promoting metastasis of osteosarcoma, and the underlying regulatory processes that control Notch pathway expression and activity in the disease. We have shown that, compared to normal human osteoblasts and non-metastatic osteosarcoma cell lines, osteosarcoma cell lines with the ability to metastasize have higher levels of Notch 1, Notch 2, the Notch ligand DLL1 and the Notch-induced gene Hes1. When invasive osteosarcoma cells are treated with small molecule inhibitors of gamma-secretase, which blocks Notch activation, invasiveness is abrogated. Direct retroviral expression has shown that Hes1 expression was necessary for osteosarcoma invasiveness and accounted for the observations. In a novel orthotopic murine xenograft model of osteosarcoma pulmonary metastasis, blockade of Hes1 expression and Notch signaling eliminated spread of disease from the tibial primary tumor. In a sample of archival human osteosarcoma tumor specimens, expression of Hes1 mRNA was inversely correlated with survival (n=16 samples, p=0.04). Expression of the microRNA 34 cluster, which is known to downregulate DLL1, Notch 1 and Notch 2, was inversely correlated with invasiveness in a small panel of osteosarcoma tumors, suggesting that this family of microRNAs may be responsible for regulating Notch expression in at least some tumors. Further, exposure to valproic acid at therapeutic concentrations induced expression of Notch genes and caused a 250-fold increase in invasiveness for non-invasive cell lines, but had no discernible effect on those lines that expressed high levels of Notch without valproic acid treatment, suggesting a role for HDAC in regulating Notch pathway expression in osteosarcoma. These findings show that the Notch pathway is important in regulating osteosarcoma metastasis and may be useful as a therapeutic target. Better understanding of Notch's role and its regulation will be essential in planning therapies with other agents, especially the use of valproic acid and other HDAC inhibitors.