Runt-related transcription factor 1 (RUNX1) is required for definitive hematopoiesis; however, the functions of most human RUNX1 isoforms are unclear. In particular, the effects of RUNX1-205 (a novel splice variant that lacks exon 6 in comparison with RUNX1b) on human hematopoiesis are not clear. In this study, a human embryonic stem cell (hESC) line with inducible RUNX1-205 overexpression was established. Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+ cells, and the production of hematopoietic stem/progenitor cells was significantly reduced. In addition, the expression of hematopoiesis-related factors was downregulated. However, these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells, indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells, while the promotive effect could be observed during the late stage of hematopoiesis. This is very similar to that of RUNX1b. Interestingly, the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b, and the protein stability of RUNX1-205 was much higher than that of RUNX1b. Thus, the function of RUNX1-205 in normal and diseased models should be further explored.