Work from our group and others has implicated Pbx homeodomain transcription factors in myocardial differentiation and outflow tract development. However, the mechanisms underlying Pbx function in heart development are not understood, and it is not known whether Pbx factors causally contribute to human congenital heart defects. We have used the CRISPR-Cas9 system to engineer zebrafish strains for a variety of pbx gene alleles to determine the roles of Pbx factors in heart development. First, we have used null alleles of the pbx2 and pbx4 genes to show that zebrafish embryos lacking both pbx2 and pbx4 show severe defects in early myocardial differentiation, myocardial morphogenesis, and outflow tract formation. Second, we have engineered in-frame deletions within the pbx4 gene to demonstrate critical functions in heart development for a domain conserved in Pbx proteins. Third, we have engineered a zebrafish strain carrying a Pbx gene variant that has been associated with congenital heart defects in human patients. We are using this strain to identify causative effects of this variant on heart and outflow tract development. Our zebrafish pbx gene alleles now allow us to test whether Pbx proteins function through transcriptional activation or repression in myocardial differentiation. Our studies provide novel examples of using genome engineering for structure-function analyses of critical heart transcription factors as well as for testing human variant functions in heart development.