We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, is important in embryonic stem (ES) cells during neurogenesis. In that study, we also observed the altered expression of mesodermal marker genes, which indicated that JSAP1 is involved in the differentiation of mesodermal lineages. Here, we investigated the function of JSAP1 in cardiomyocyte development using JSAP1-null ES cells, and found that cardiomyogenesis was impaired in the JSAP1-null mutant. The JSAP1 deficiency resulted in lower gene expression of the cardiac transcription factor Nkx2.5 and contractile proteins. In contrast, the mutant showed a significantly higher expression of mesoderm-related markers other than those of the cardiomyocyte lineage. Together, these results suggest that JSAP1 may be important for the differentiation of the mesodermal lineages, functioning as a positive factor for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into other lineages.