Role of autophagy in the maintenance of definitive hematopoiesis using zebrafish platform Autophagy is a dynamic and evolutionary conserved lysosomal degradation pathway for cellular remodeling, development and homeostasis. It is also essential for the maintenance of different hematopoietic lineages including erythroid, myeloid, and lymphoid ones, yet its function in definitive hematopoiesis is not clear. Here, we inhibited autophagy in zebrafish (Danio rerio) embryos via ulk1b (homologous to human ULK1) knock-out to observe hematopoiesis. Taken advantages of the optically clear and externally fertilized zebrafish embryos together with the genetic tractability and pharmacological approaches, here we reported that, zebrafish lacking the key autophagy gene ulk1b inhibit autophagy at the earlier stage by blocking the autophagosome formation or activation process. Dysfunction of the ulk1b gene in zebrafish embryos resulted in minimal cytosolic microtubuleassociated protein 1A/1B-light chain 3 (Lc3I) conjugation to phosphatidylethanolamine (PE) to form LC3-PE conjugate (LC3-II) followed by decreased autophagosomal membrane recruitment. Consequently, in-vivo live imaging showed that inhibition of autophagy further suppress autophagosomes and autolysosomes numbers. However, this autophagy inhibition modulated the hematopoietic lineage specific concomitant upregulation of myeloid progenitors (spi1b), pan-leukocytes (lcp1), macrophages (mpeg1.1) and neutrophils (mpo). Conversely, erythroid progenitors (gata1), embryonic hemoglobin (hbae1.1) and hematopoietic stem cell (cmyb) numbers significantly decreased. Treatment with autophagy inducer calpeptin was not sufficient to ameliorate the defective hematopoiesis in mutant embryos. These findings indicated that lack of autophagy throughout definitive hematopoiesis incorporate with myeloproliferation and anemia in zebrafish embryos which further warrant important role of autophagy to maintain normal HSC functions.