Differentiation-inducing factors, produced by Dictyostelium discoideum, show anti-tumor activity by suppressing the Wnt/β-catenin signaling pathway via activation of GSK-3 in several human tumor cells. We tried to clarify the all signaling pathways affected by DIF using DNA microarray analysis and found that Hippo signaling pathway is one of pathways which activities were modified by DIFs. The Hippo signaling pathway controls organ size in animals through the regulation of cell proliferation and apoptosis. Further, Hippo signaling pathway is known to be related with cancer development and metastasis formation. DIF-1 and DIF-3 significantly elevated expression of Hippo signaling pathway target genes in a time-dependent manner. We next analyzed the effect of DIF-1 on Yes-associated protein (YAP), a key transcriptional coactivator in the Hippo signaling pathway. We found that DIF-1 significantly reduced phosphorylation level of YAP, thereby activating YAP to translocate to nucleus in HeLa cells. We now try to clarify the mechanism by which DIF reduces YAP phosphorylation.