AbstractThe Notch pathway is an intercellular signaling mechanism frequently used for controlling cell fate during organogenesis. There are four structurally related Notch receptors in mice and humans, and Notch1 and Notch2 are essential genes. In this report we describe the construction of a transgenic mouse strain that expresses the Notch2 intracellular domain in response to cell lineage specific expression of Cre recombinase. This approach bypasses the requirement for ligand‐ receptor interaction and allows the direct determination of the consequences of Notch2 activation in vivo. Exogenous expression of the Notch2 intracellular domain resulted in the developmental arrest of secondary heart field derived cardiomyocytes during the transition from immature α‐Smooth Muscle Actin expressing cells to mature α‐Actinin positive cardiomyocytes. In contrast, a cell nonautonomous mesenchymal expansion was observed in semilunar valves. This new conditionally expressed allele of Notch2 can be used in studies by investigators interested in the effects of Notch2 activation in vivo. genesis, 47:573–578, 2009. Published 2009 Wiley‐Liss, Inc.