Pea3 and Erm are transcription factors expressed in normal developing branching organs such as the mammary gland. Deregulation of their expression is generally associated with tumorigenesis and particularly breast cancer. By using RNA interference (RNAi) to downregulate the expression of Pea3 and/or Erm in a mammary cancer cell line, we present evidence for a role of these factors in proliferation, migration and invasion capacity of cancer cells. We have used different small interfering RNAs (siRNAs) targeting pea3 and erm transcripts in transiently or stably transfected cells, and assessed the physiological behavior of these cells in in vitro assays. We also identified an in vivo alteration of tumor progression after injection of cells that overexpress pea3 and/or erm short hairpin RNAs (shRNAs) in immunodeficient mice. Using transcriptome profiling in Pea3- or Erm-targeted cells, two largely independent gene expression programs were identified on the basis of their shared phenotypic modifications. A statistically highly significant part of both sets of target genes had previously been already associated with the cellular signaling pathways of the ;proliferation, migration, invasion' class. These data provide the first evidence, by using endogenous knockdown, for pivotal and complementary roles of Pea3 and Erm transcription factors in events crucial to mammary tumorigenesis, and identify sets of downstream target genes whose expression during tumorigenesis is regulated by these transcription factors.