Immunoreceptor tyrosine-based activation motifs (ITAMs) are involved in the transduction of signals necessary for activation, differentiation, and survival in hematopoietic cells. Several viruses have been shown to encode ITAM-containing transmembrane proteins. Although expression of these viral proteins has in some cases been shown to transform nonhematopoietic cells, a causal role for a functional ITAM in this process has not been elucidated. To examine the potential transforming properties of ITAM-containing proteins, a recombinant protein consisting of ITAM-containing cytoplasmic regions of the B-cell antigen receptor was expressed in immortalized murine mammary epithelial and fibroblast cells. Mammary epithelial cells expressing this construct exhibited depolarized morphology in three-dimensional cultures. This transformed phenotype was characterized by a loss of anchorage dependence and hallmarks of epithelial to mesenchymal transition. Fibroblasts expressing this ITAM construct also lost contact inhibition and anchorage dependence. The transformed phenotype seen in both cell types was abrogated upon tyrosine to phenylalanine substitutions of the ITAMs. Inhibition of Syk tyrosine kinase, which associates with the ITAM, also prevented cell transformation. Our results indicate that expression of a nonviral ITAM-containing protein is sufficient for cell transformation. Despite lacking intrinsic enzymatic activity, ITAM-containing proteins can function as potent oncoproteins by scaffolding downstream mediators.