Hemangioblast, including primitive hematopoietic progenitor cells, play an important role in hematopoietic development, however, the underlying mechanism for the propagation of hematopoietic progenitor cells remains elusive. A variety of regulatory molecules activated in early embryonic development play a critical role in the maintenance of function of hematopoietic progenitor cells. Homeobox transcription factors are an important class of early embryonic developmental regulators determining hematopoietic development. However, the effect of homeobox protein Hox-B4 (HOXB4) ectopic expression on the development of hemangioblasts has not been fully addressed. This study aimed to investigate the role ofHoxb4a, an ortholog gene ofHOXB4in zebrafish, in the hematopoietic development in zebrafish. A transgenic zebrafish line was established withCre-loxPsystem that stably overexpressed enhanced green fluorescent protein (EGFP)-tagged Hoxb4a protein under the control of hemangioblast-specificlmo2promoter. Overexpression ofHoxb4ain the development of hemangioblasts resulted in a considerable increase in the number ofstem cell leukemia (scl)andlmo2-positive primitive hematopoietic progenitor cells occurring in the posterior intermediate cell mass (ICM). Interestingly,Hoxb4aoverexpression also disrupted the development of myelomonocytes in the anterior yolk sac and the posterior ICM, without affecting erythropoiesis in the posterior ICM. Taken together, these results indicate thatHoxb4afavours the development of hematopoietic progenitor cells originated from hemangioblastsin vivo.