<p>Studying the mechanism of hematopoietic stem cells’ generation from induced pluripotent stem cells in vitro can be useful for understanding embryonic hematopoiesis, as well as for the application of related cell therapy. This study aimed to delineate the process of the differentiation of induced pluripotent stem cells into hematopoietic stem cells’ models and provide a theoretical basis and clinical value for the production of hematopoietic stem cells in vitro. We analyzed the differentiation model by single-cell dynamic transcriptome and glycosylation sequencing, which was divided into three differentiation stages based on the new‐to‐total RNA ratio and glycosylation level. Two differentiation fates were found in the pseudo-time, including hematopoietic development and other tissue development. Precursor hematopoietic cells with a high glycosylation level greatly expressed hematopoietic regulation and vascular endothelial genes, suggesting that glycosylation is associated with angiogenesis and hematopoietic regulation. The multiple differentiation events in the in vitro model are similar to those in hematopoietic development in vivo, including yolk sac hematopoiesis, cellular communication between non-potential hematopoietic subsets and potential hematopoietic subsets, gene expression, and temporal deviations in hematopoietic fate. Our study has revealed the similar hematopoiesis process in the differentiation model via single-cell dynamic RNA and glycosylation sequencing, which provides an important theoretical basis for the study of hematopoietic stem cell development.</p>