The P19 embryonic stem cell line is a useful model system for studies of cardiac differentiation. Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain pluripotency of ES cells. However, the molecular mechanism through which nanog regulates stem cell pluripotency remains unknown. In order to identify transcription factors involved in early cardiac differentiation from P19 cells, we analyzed the mRNA expression profiles by cDNA microarray in P19 cells transfected with short interfering RNAs (siRNAs) against nanog. Total RNAs were extracted from P19 cells transfected with the nanog-siRNA-duplexes selected from three different target regions of nanog cDNA, hybridized to microarrays which represent approximately 14,000 cDNAs and expressed sequence tags (ESTs), and results were analyzed using the significance analysis of microarrays (SAM) method. The nanog-siRNA-100 duplexes effectively decreased the expression of nanog up to 23.8% than other two kinds of siRNAs, the nanog-siRNA-400 (68.3 %) and -793 (53.1%) as determined by real-time RT-PCR. Transcript profiling revealed differential expression a total of 187 genes (142 increased; 45 decreased) which showed a factor of 2-fold or greater change in the nanog-siRNA-100 transfected P19 cells compared to control cells. Among 187 genes, 65 were ESTs or were unknown function, and the remain 122 genes had known functions mainly relating to cell cycle, stem cell maintenance and differentiation, embryo development, signal transduction, transcriptional regulation. Specifically, genes which may be involved in stem cell maintenance and cardiac differentiation such as Oct-4, frizzled homolog 4, BCL2-associated transcription factor 1, tubby like protein 4, zinc finger proteins, leucine-rich factors, fork-head box factors, novel ESTs were further analyzed. Real-time RT-PCR analysis validated that transcript levels of those molecules were identical to cDNA array data. The functional roles of selected genes during cardiac differentiation have been undertaken in P19 cells. Genes identified in this study are promising candidates for involvement in the cardiac differentiation of embryonic stem cells.