Thrombopoietin (TPO), a cytokine that participates in the differentiation and maturation of megakaryocytes, is produced in the liver, but only limited information is available on the biologic response of liver-derived cells to TPO. In this study we investigated whether HepG2 express c-mpl, the receptor for TPO, and whether TPO elicits biologic responses and intracellular signaling in this cell type. Specific transcripts for c-mpl were detected in HepG2 by RT-PCR, and expression of the protein was demonstrated by western blotting and immunofluorescence. Exposure of HepG2 to TPO was associated with a dose-dependent increase in cell migration and chemo-invasion through Matrigel-coated filters. A checkerboard analysis showed that the effects of TPO on migration were dependent on both chemotaxis and chemokinesis. Exposure of HepG2 to TPO resulted in the activation of different members of the MAPK family, including ERK and JNK, as assessed by using phosphorylation-specific antibodies and immune complex kinase assays. TPO also activated phosphatidylinositol 3-kinase (PI-3K) and the downstream kinase Akt in a time-dependent manner. Finally, activation of c-mpl was associated with increased activation of nuclear factor-B. Using specific inhibitors, tyrosine phosphorylation, and activation of PI-3K were found to be required for the induction of migration in response to TPO. We conclude that TPO exerts biological actions on cultured hepatoblastoma cells via activation of c-mpl and its downstream signaling.