Expression of the alpha6beta4 integrin is associated with poor patient prognosis and reduced survival in a variety of human cancers. In recent years, a limited number of in vivo studies have examined the contribution of this integrin receptor to cancer progression and they have revealed that the alpha6beta4 integrin plays a multifaceted role in regulating tumor development and progression. In the current study, we investigated the mechanism by which one tyrosine residue in the beta4 subunit cytoplasmic domain, Y1494, contributes to the tumor-promoting functions of the alpha6beta4 integrin in vivo. We show that Y1494 participates in the stimulation of diverse signaling pathways that promote alpha6beta4-dependent tumor growth and invasion. Mutation of Y1494 inhibits the ability of the alpha6beta4 integrin to support anchorage-independent growth in vitro and tumor development and angiogenesis in vivo, a result that mimics the loss of total expression of the beta4 subunit. Our results support the hypothesis that Y1494 regulates alpha6beta4-dependent anchorage-independent growth through activation of the extracellular signal-regulated kinase 1/2 signaling pathway, and invasion through the combined activation of phosphatidylinositol 3-kinase and Src. Collectively, our results identify Y1494 as a major regulatory site for signaling from the alpha6beta4 integrin to promote tumor development and progression.