Objective: Fetal inactivation of the cardiac transcription factors Gata4 and Gata6 has been shown to cause postnatal heart dysfunction. However, the postnatal requirement for these transcription factors has not been defined. Methods: Using systemic delivery of recombinant adeno-associated virus 9 expressing Cre from the cardiac specific TNNT2 promoter (1.2E x 12VG/gram body weight; n=6), we inactivated floxed Gata4 and Gata6 alleles in neonatal or adult hearts. Left ventricular performance was followed serially using M-mode echocardiography and two-dimensional speckle tracking-derived myocardial tissue deformation (Visualsonics Vevo 2100) under light isofluorane anesthesia. The intervention group was compared to a control AAV-TNNT2 encoding luciferase. Findings: In the neonatal heart (P1-P5), effective knock-down of Gata4 and Gata6 (n=6 mice) was confirmed by Western blot and quantitative reverse-transcription PCR of myocardial tissue: We observed a 4.9-fold reduction in Gata4 expression; (95% C.I. +/-1.2) and a 6-fold reduction in Gata6 expression (95% C.I. +/- 1.2). Over the course of 5 days, left ventricular ejection fraction decreased significantly from a mean of 81% to 39% +/- 3% SEM (p<0.0001). Strain analysis confirmed a global reduction in myocardial performance after knock-down. The expression of heart failure markers, m-ANF and MYH7, were markedly up-regulated at 17-fold and 24-fold, respectively. In the adult heart (postnatal weeks 6-9, n=6), despite a technically successful systemic delivery of rAAV9, no noted left ventricular dysfunction was apparent on subsequent serial follow-up by m-mode echocardiography. Similarly, myocardial performance, as measured by strain & strain rate analysis in the longitudinal, circumferential and radial planes, remained equal in the intervention group to that of the control group. Conclusions: AAV9-TNNT2-Cre is an effective means of achieving temporally controlled inactivation of floxed alleles in the postnatal heart. Gata4 and Gata6 are required to maintain normal left ventricular systolic function during neonatal life, but are no longer required in adulthood under normal physiological conditions.