The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during mouse embryonic development. Consequently, Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. Previously we found that mutating the only runx1 homologue in zebrafish also resulted in deficiency of definitive blood cells during embryogenesis. However, the zebrafish runx1 mutants, which carried an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X), were able to recover from a larval “bloodless” phase and develop to fertile adults with multi-lineage hematopoiesis (Sood et al., Blood 2010), suggesting that definitive hematopoiesis can develop without RUNX1. To confirm this finding and to further study the underlying mechanism, we generated three new runx1 mutants: two mutations in exon 4 of runx1 (a deletion of 8 bp, runx1del8/del8, and a deletion of 25 bp, runx1del25/del25) that truncate the runt-homologous domain; and a large deletion of exons 3 through 8 (runx1del(e3-8)/del(e3-8)), which removes most coding region of runx1, using the TALEN and CRISPR-Cas9 technologies. All three new runx1 mutants failed to develop definitive hematopoiesis during early embryonic development. Time-lapse recordings with confocal microscopy revealed no emergence of c-myb+ HSCs from the ventral wall of dorsal aorta in the runx1 mutant embryos at 48 hours post fertilization. However, a second wave of HSCs emerged that led to circulating blood cells between 15 and 20 days post fertilization. Eventually, about 40% of the runx1 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data strongly suggests for a RUNX1-independent mechanism for HSC formation and definitive hematopoiesis.