Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt.