Notch signaling has been shown to mediate a wide array of cell fate decisions during development. While previous work has demonstrated that Notch signaling plays an important role in regulating cardiac differentiation and morphogenesis, an earlier role during cardiac field formation has not yet been fully characterized. Previously, our lab demonstrated that perturbations in Notch signaling beginning at the onset of gastrulation affect the subdivision of germ layers. However due to the potential additive effects of misregulating Notch signaling over multiple stages of development, it was not possible to distinguish a specific role for this pathway during heart field specification. Here, we developed an innovative approach that takes advantage of temporally inducible constructs to isolate our manipulations to specific windows of development. In particular, we focused our studies on some of the earliest stages of cardiogenesis when heart field specification occurs. Our findings demonstrate a novel role for Notch signaling during the prepatterning of the cardiac mesoderm. Specifically, once relieved of aberrantly activated Notch signaling following gastrulation, cardiac precursors retain the ability to express markers of the cardiac field. Conversely, downregulating Notch signaling in cells fated to become heart tissue results in the induction of cardiac field genes in gastrula embryos. Finally, we provide evidence suggesting that this new role for Notch signaling is mediated at least in part via the Notch effector protein, Esr9 and the transcription factor GATA4. Taken together, these findings provide strong evidence for a novel role for Notch signaling in regulating the timing of heart field specification during early cardiogenesis.