Lysine-specific Demethylase 1B (KDM1B) is a chromatin regulator which functions as a histone eraser through the removal of the post-translational modifications mono and dimethylation of histone 3 on lysine 4 (H3K4me1/2). This process is enhanced by the formation of a complex with Nuclear Protein Glyoxylate Reductase (NPAC). NPAC resolves the sequestration of the nucleosome histone tail to allow robust demethylation of H3K4me2 by KDM1B, during transcriptional elongation by RNA polymerase 2 (RNAP II). KDM1B is involved in many crucial processes during development. Its physiological functions include the establishment of maternal genomic imprints, reset of the epigenome during somatic cell reprogramming, and regulation of brown adipogenic differentiation. In light of this, the role of KDM1B in human embryonic stem cells (hESCs) is examined through CRISPR/Cas9-editing to further dissect its biological functions during embryogenesis. CRISPR-induced knockouts of KDM1B exhibited similar cell proliferation rate and expression of OCT4 and NANOG pluripotency markers to wildtype cells. Furthermore, KDM1B-/- clones were able to maintain their pluripotency potential by differentiating to all germ layers in teratoma and embryoid body formation assays. In addition, RNA-seq of KDM1B-/- clones showed enrichment of mesoderm lineage-related gene ontology (GO) terms in the downregulated differentially expressed genes. Thus, KDM1B is believed to be dispensable during the pluripotent stage of the cell but proved fundamental during later stages of development.