Granulocyte colony-stimulating factor receptor (GCSFR) signaling participates in the production of neutrophilic granulocytes during normal hematopoietic development, with a particularly important role during emergency hematopoiesis. This study describes the characterization of the zebrafish gcsf and gcsfr genes, which showed broad conservation and similar regulation to their mammalian counterparts. Morpholino-mediated knockdown of gcsfr and overexpression of gcsf revealed the presence of an anterior population of myeloid cells during primitive hematopoiesis that were dependent on GCSF/GCSFR for their development and migration. This contrasted with a posterior domain that was largely independent of this pathway. Definitive myelopoiesis was also partially dependent on a functional GCSF/GCSFR pathway. Injection of bacterial lipopolysaccharide elicited significant induction of gcsf expression and emergency production of myeloid cells, which was abrogated by gcsfr knockdown. Collectively, these data demonstrate GCSF/GCSFR to be a conserved signaling system for facilitating the production of multiple myeloid cell lineages in both homeostatic and emergency conditions, as well as for early myeloid cell migration, establishing a useful experimental platform for further dissection of this pathway.