The transcription factor orthodenticle homeobox 2 (OTX2) has been implicated in the pathogenesis of medulloblastoma, as it is often highly expressed and sometimes amplified in these tumors. Little is known of the downstream pathways regulated by OTX2. We therefore generated MED8A and DAOY medulloblastoma cell lines with doxycycline-inducible OTX2 expression. In both cell lines, OTX2 inhibited proliferation and induced a senescence-like phenotype with senescence-associated β-galactosidase activity. Expression profiles of time series after OTX2 induction in MED8A showed early upregulation of cell cycle genes related to the G2-M phase, such as AURKA, CDC25C, and CCNG2. Paradoxically, G1-S phase genes such as MYC, CDK4, CDK6, CCND1, and CCND2 were strongly downregulated, in line with the observed G 1 arrest. ChIP-on-chip analyses of OTX2 binding to promoter regions in MED8A and DAOY showed a strong enrichment for binding to the G2-M genes, suggesting a direct activation. Their mRNA expression correlated with OTX2 expression in primary tumors, underscoring the in vivo relevance of this regulation. OTX2 induction activated the P53 pathway in MED8A, but not in DAOY, which carries a mutated P53 gene. In DAOY cells, senescence-associated secretory factors, such as interleukin-6 and insulin-like growth factor binding protein 7, were strongly upregulated after OTX2 induction. We hypothesize that the imbalance in cell cycle stimulation by OTX2 leads to cellular senescence either by activating the P53 pathway or through the induction of secretory factors. Our data indicate that OTX2 directly induces a series of cell cycle genes but requires cooperating genes for an oncogenic acceleration of the cell cycle. ©2010 AACR.