The generation of hematopoietic stem cells from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. Achieving this goal is complicated by our incomplete understanding of the mechanism regulating definitive hematopoietic specification. We used our stage-specific hPSC differentiation method to obtain and identify, via CD235a expression, mesoderm harboring exclusively primitive or definitive hematopoietic potential to understand the genetic regulation of definitive hematopoietic specification. Whole-Transcriptome gene expression analyses on WNT-dependent KDR1 CD235a2 definitive hematopoietic mesoderm and WNT-independent KDR1CD235a1 primitive hematopoietic mesoderm revealed strong CDX gene expression within definitive hematopoieticmesoderm. Temporal expression analyses revealed that CDX4 was expressed exclusively within definitive hematopoietic KDR1CD235a2 mesoderm in a WNT-and fibroblast growth factor-dependent manner. We found that exogenous CDX4 expression exclusively during mesoderm specification resulted in a >90% repression in primitive hematopoietic potential, but conferred fivefold greater definitive hematopoietic potential, similar to that observed following WNT stimulation. In contrast, CDX4 knockout hPSCs had intact primitive hematopoietic potential, but exhibited a fivefold decrease in multilineage definitive hematopoietic potential. Taken together, these findings indicate that CDX4 is a critical transcription factor in the regulationofhumandefinitivehematopoieticspecification,andprovides amechanisticbasis forWNT-mediateddefinitivehematopoietic specification from hPSCs.