Cell-surface polysaccharides are a fundamental component of the stem cell microenvironment. They are known to modulate developmental signals- critical for pluripotency and differentiation. Nevertheless, the architecture of the cellular glycocalyx and how these structures direct the fate of human pluripotent stem (hPS) cells have not been fully explored. I addressed this gap with a focus on a critical glycan of the hPS cells niche, heparan sulfate (HS). HS is a heterogeneous long-chain cell-surface polysaccharide. The spatial distribution and ultrastructure of this information-rich, signaling polysaccharides are poorly defined. In this work, I aim to understand the interplay between the HS organization and the developmental signal transduction in the hPS cells’ microenvironment. We discovered that HS of hPS cells has a dynamic ultrastructure that undergoes changes during lineage-specific differentiation. These changes also correlate with the cells’ ability to bind specific growth factor. While variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our findings indicate a role for HS ultrastructure in its ability to recruit growth factors in stem cell niche. To advance the understanding of its roles in human development, next we engineered a HS-deficient cell line derived from hPS cells. Parallelly, I set out to develop a synthetic, modular surface-based cell separation strategy that can isolate or enrich cells of interest in a rapid and label-free way. I applied this strategy to isolate genetically engineered HS-deficient hPS cells after a CRISPR modification by engaging the cell surface HS with a small peptide- presenting synthetic surface. These HS-deficient hPS cells aid the investigation further to understand the role of HS in human development. We showed that the multi-lineage commitment of hPS cells depends on HS. Moreover, lack of HS hinders the proper neuronal projections and synaptic vesicle formation in hPS cell-derived neurons, suggesting a specific role of HS in human neural development. Taken together, these results indicate that HS has a highly dynamic ultrastructure that modulates cell fate choices of hPS cells, specifically neuronal connection formation. This work paves the way to a better understanding of HS’s role in early human development.Ph.D.