Valve formation during embryonic heart development involves a complex interplay of regional specification, cell transformations, and remodeling events. While many studies have addressed the role of specific genes during this process, a global understanding of the genetic basis for the regional specification and development of the heart valves is incomplete. We have undertaken genome-wide transcriptional profiling of the developing heart valves in the mouse. Four Serial Analysis of Gene Expression libraries were generated and analyzed from the mouse atrio-ventricular canal (AVC) at embryonic days 9.5-12.5, covering the stages from initiation of endothelial to mesenchymal transition (EMT) through to the beginning of endocardial cushion remodeling. We identified 14 distinct temporal patterns of gene expression during AVC development. These were associated with specific functions and signaling pathway members. We defined the temporal distribution of mesenchyme genes during the EMT process and of specific Notch and transforming growth factor-beta targets. This work provides the first comprehensive temporal dataset during the formation of heart valves. These results identify molecular signatures that distinguish different phases of early heart valve formation allowing gene expression and function to be further investigated.