Prior work has indicated that D-type cyclin–cdk4 complexes, which are only active in proliferating cells, can suppress the skeletal muscle differentiation program in proliferating myoblasts. In this study, we show that cyclin D–cdk activity can block the activity of the MEF2 family of transcriptional regulators, which are crucial regulators of skeletal muscle gene expression. We have found that cyclin D–cdk activity blocks the association of MEF2C with the coactivator protein GRIP-1 and thereby inhibits the activity of MEF2. During skeletal muscle differentiation, GRIP-1 is localized to punctate nuclear structures and can apparently tether MEF2 to such structures. Cotransfection of GRIP-1 can both potentiate the transcriptional activity of a Gal4–MEF2C construct and induce MEF2C localization to punctate nuclear structures. Consistent with the absence of punctate nuclear GRIP-1 in proliferating myoblasts, we have found that ectopic cyclin D–cdk4 expression disrupts the localization of both GRIP-1 and MEF2C to these punctate subnuclear structures. Our findings indicate that cyclin D–cdk4 activity represses skeletal muscle differentiation in proliferating cells by blocking the association of MEF2 with the coactivator GRIP-1 and concomitantly disrupts the association of these factors with punctate nuclear subdomains within the cell.