GATA5 is a member of the zinc finger transcription factor GATA family (GATA1-6) that plays a wide variety of roles in embryonic and adult development. Experiments in multiple model systems have emphasized the importance of the GATA family members 4 -6 in the development of the endoderm and mesoderm. Yet despite overlapping expression patterns, there is little evidence of an important role for GATA5 in mammalian cardiac development. We have generated a new Gata5 mutant allele lacking exons 2 and 3 that encodes both zinc finger domains (Gata5 tm2Eem ), and we show that although Gata5 All six members of the GATA family (GATA1-6) of zinc finger transcription factors play important roles in cell fate decision, differentiation, and morphogenesis (1). Members of this family recognize the GATA motif, which is present in the promoters of many genes. These factors have been divided into two subfamilies, Gata1/2/3 and Gata4/5/6, based on their expression patterns and amino acid sequence homology (2). Gata1, Gata2, and Gata3 are preferentially expressed in hematopoietic cells and regulate proliferation and differentiation during hematopoiesis (3). Gata4, Gata5, and Gata6 are predominantly expressed during embryonic heart development, in addition to other sites. The DNA binding domains of GATA4, GATA5, and GATA6 are ϳ85% similar at the protein level. During early cardiac development, Gata4 expression is confined to the cardiac crescent, and later the transcripts are detected throughout the myocardium and endocardium (4, 5). Gata4-deficient mice die between E8.5 4 and E10.5 because of defects in ventral morphogenesis, including a failure of the cardiac mesoderm to form a linear heart tube (6, 7). A frameshift mutation in human GATA4 (E359del) is linked to cardiac septal defects (8). Like Gata4, Gata6 is expressed in the precardiac mesoderm and later in myocardial cells. Gata6 is also expressed in vascular smooth muscle (9). Gata6 null mice die prior to cardiac development because of defects in the visceral endoderm function and extraembryonic development (10, 11).The temporal and spatial expression pattern of Gata5 suggests its involvement in tissue-specific transcriptional regulation during embryonic development (12). Gata5 is expressed in the precardiac crescent between E7 and E8 (12). By E9.5 in the heart, Gata5 is expressed at high levels in the atria with lower levels observed in the ventricle and outflow tract. By E12.5, expression is primarily restricted to endocardial cells of the atria, and by E16.5, Gata5 transcripts are no longer detected in cardiac tissues (12). In chick, GATA5 is transcribed in the cardiac crescent prior to formation of the primordial heart tube (13). In Xenopus, gata5 is expressed in both cardiac mesoderm and hepatogenic endoderm. Down-regulation of gata5 using two nonoverlapping translation-blocking and splice site-blocking morpholino causes severe reduction in the number of heart and liver precursors at the time of or shortly after their specification (14). In zebrafish, gata5 is encoded by th...