ABSTRACTCollagen X is a non‐fibril collagen produced by hypertrophic chondrocytes and was believed to associate with the calcification process of growth plate cartilage. The homozygous loss ofCol10a1gene in mice, however, demonstrated no remarkable effects on growth plate formation or skeletal development. To investigate the role of collagen X in human chondrocytes, we established human induced pluripotent stem cells (hiPSCs) with heterozygous (COL10A1+/−) or homozygous (COL10A1−/−) deletions ofCOL10A1gene using the dual sgRNA CRISPR/Cas9 system. Several mutant clones were established and differentiated into hypertrophic chondrocytes by a previously reported 3D induction method. No remarkable differences were observed during the differentiation process between parental and mutant cell lines, which differentiated into cells with features of hypertrophic chondrocytes, indicating that collagen X is dispensable for the hypertrophic differentiation of human chondrocytes in vitro. To investigate the effects of collagen X deficiency in vivo, chondrocyte pellets at the proliferating or prehypertrophic stage were transplanted into immunodeficient mice. Proliferating pellet‐derived tissues demonstrated the zonal distribution of chondrocytes with the transition to bone tissues mimicking growth plates, and the proportion of bone tended to be larger inCOL10A1−/−tissues. Prehypertrophic pellet‐derived tissues produced trabecular bone structures with features of endochondral ossification, and there was no clear difference between parental‐ and mutant‐derived tissues. A transcriptome analysis of chondrocyte pellets at the hypertrophic phase showed a lower expression of proliferating‐phase genes and a higher expression of calcification‐phase genes inCOL10A1−/−pellets compared with parental cell pellets. These in vitro and in vivo data suggested that collagen X is dispensable for the hypertrophic differentiation and endochondral ossification of human iPSC‐derived chondrocytes, though it may facilitate the differentiation process. Thus,COL10A1−/−iPSC lines are useful for investigating the physiological role of collagen X in chondrocyte differentiation. © 2023 The Authors.JBMR Pluspublished by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.