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 isoform specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K and p53 in transformation. Comparisons of polyoma small T (PyST) and SV40 small T (SVST) implicate Aβ in control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulates differentiation; its activation or inhibition respectively promoted or blocked. Aβ binds Akt. Enhancement of PP2A Aβ/Akt interaction by PyST increased turnover of Akt S473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Akt S473. This provides new insight into regulation of Akt, a protein of extreme importance in cancer. Further, our results suggest that the role for Aβ in differentiation, and perhaps tumor suppression, may partly lie in its ability to negatively regulate Akt.