Growth factor independence 1 (GFI1) is important for maturation of mammalian lymphocytes and neutrophils and maintenance of adult hematopoietic stem cells (HSCs). The role of GFI1 in embryonic hematopoiesis is less well characterized. Through an enhancer trap screen and bioinformatics analysis, we identified a zebrafish homolog of Gfi1 (named gfi1.1) and analyzed its function during embryonic development. Expression of both an endogenous gfi1.1 gene and a GFP reporter gene inserted near its genomic locus was detected in hematopoietic cells of zebrafish embryos. Morpholino (MO) knockdown of gfi1.1 reduced expression of scl, lmo2, c-myb, mpo, rag1, gata1 and hemoglobin alpha embryonic-1 (hbae1), as well as the total amount of embryonic hemoglobin, but increased expression of pu.1 and l-plastin. Under the same conditions, MO injection did not affect the markers involved in vascular and pronephric development. Conversely, overexpression of gfi1.1 via mRNA injection enhanced expression of gata1 but inhibited expression of pu.1. These findings suggest that Gfi1.1 plays a critical role in regulating the balance of embryonic erythroid and myeloid lineage determination, and is also required for the differentiation of lymphocytes and granulocytes during zebrafish embryogenesis.