In order to determine the importance of hhlim in cardiomyocyte differentiation and to define the hierarchy of transcription factors during cardiac development, the cardiac differentiation profile of the pluripotent P19 cells was characterized at cellular and molecular level. Genes that express early in the cardiogenesis of the embryo include GATA-4 and Nkx2.5. The time course of expression of these genes during in vitro differentiation of P19 cells into cardiomyocytes was established using RT-PCR. GATA-4 is expressed at day 2 after treatment with DMSO, and Nkx2.5 at day 3. hhlim is almost undetected until day 4 after DMSO treatment. Western blot had the same results, which showed that hhlim might participate in the cardiac differentiation pathway. P19-hhlim-overexpressing cells did not show morphological or biochemical evidence of cardiomyogenesis without DMSO treatment, but overexpression of hhlim had a benefit effect on cell aggregates that were especially obvious in the absence of DMSO. Moreover, when hhlim-overexpressing cells were treated with DMSO, the expression of Nkx2.5 and GATA-4 appeared earlier compared with the control. hhlim-deficient P19 cells induced by DMSO retained the ability to differentiate into cardiomyocytes. The time course of expression of Nkx2.5 and GATA-4 in the hhlim-deficient clones was significantly reduced, whereas these clones treated by RA produced neuroectodermal derivatives. Then it was concluded that lack of hhlim expression specifically inhibits cardiomyocyte formation of P19 cells without affecting neuronal differentiation pathway. These data showed that overexpression of hhlim could enhance cardiogenesis of embryonic stem cells whose mechanism might be involved in upregulation of the expression of Nkx2.5 and GATA-4.