To study the transcriptome of embryonic stem cells, we used a new gene expression profiling method that can measure the expression levels of unknown and rarely expressed transcripts precisely. We detected a total of 33,136 signal peaks representing transcripts in mouse embryonic stem cells, E14. Subsequent random cloning of the peaks suggests that mouse embryonic stem cells express at least 40,000 transcripts, of which about 2,000 are still unknown. In addition, we identified 1,022 noncoding transcripts, several of which change depending on differentiation in gene expression. Our database provides a high-resolution expression profile of E14 cells and is applicable to other mouse embryonic stem cell analyses. It includes most transcription regulation factor-encoding genes and a significant number of unknown and noncoding transcripts.