The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been associated with a variety of cellular functions ranging from cell cycle regulation to tissue development. Although years of research have extensively characterized this signaling pathway, little is known as to how specific cellular events are coordinated by its activation. Here we demonstrate that Dapr (differentiation-associated protein), a novel protein, appears to focus one aspect of this pathway by acting as a putative scaffold protein during skeletal muscle differentiation. We present for the first time a description of this protein using in silico analysis. dapr was discovered through a previous study employing chromatin immunoprecipitation and CpG microarray analysis experiments as being regulated by myocyte-enhancing factor 2, a key transcription factor involved in the differentiation of skeletal muscle tissue. In this study we show that during the course of differentiation, Dapr binds to the PI3K signaling pathway member protein kinase B (PKB). In C2C12 myoblast cells before differentiation Dapr is localized to the cytosol, migrating with PKB to the membrane after initiation of muscle differentiation. Knockdown of Dapr by RNAi resulted in inhibition of myotube formation. Our findings indicate that Dapr is a key component required by myoblasts for orchestrating their differentiation during myogenesis. Furthermore, it appears that Dapr is involved in the PI3K signaling cascade, potentially acting as a scaffold protein for PKB and coordinating its compartmentalization during differentiation.