Growth Factor Independence (Gfi) transcription factors play essential roles in 
hematopoiesis, differentially activating and repressing transcriptional programs 
required for hematopoietic stem/progenitor cell (HSPC) development and lineage 
specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), 
myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, 
megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for 
primitive hematopoiesis; however, little is known about the role of gfi1aa in 
definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we 
report the isolation and characterization of an additional hematopoietic gfi 
factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and 
definitive sites of hematopoiesis in zebrafish. Our functional analyses 
demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive 
and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences 
markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b 
silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for 
definitive hematopoiesis. We determine the epistatic relationships between the 
gfi factors and key hematopoietic transcription factors, demonstrating that 
gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of 
teleost HSPC. Our studies establish a comparative paradigm for the regulation of 
hematopoietic lineages by gfi transcription factors.