The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin-dependent kinase 9 (Cdk9), a core component of positive elongation factor-b, is a novel GATA4-binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac-specific Nkx2.5 promoter, were induced to differentiate on feeder-free gelatin-coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP-positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant-negative form of Cdk9 (DN-Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6-dichloro-1-β-ribofuranosyl-benzimidazole (DRB) on TSA-induced myocardial cell differentiation. The introduction of the DN-Cdk9 inhibited TSA-induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA-induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4. J. Cell. Physiol. 226: 248–254, 2010. © 2010 Wiley-Liss, Inc.