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Epidemiological studies suggest tobacco consumption as a probable environmental factor for a variety of congenital anomalies, including low bone mass and increased fracture risk. Despite intensive public health initiatives to publicize the detrimental effects of tobacco use during pregnancy, approximately 10–20% of women in the United States still consume tobacco during pregnancy, some opting for so-called harm-reduction tobacco. These include Snus, a type of orally-consumed yet spit-free chewing tobacco, which is purported to expose users to fewer harmful chemicals. Concerns remain from a developmental health perspective since Snus has not reduced overall health risk to consumers and virtually nothing is known about whether skeletal problems from intrauterine exposure arise in the embryo.Utilizing a newly developed video-based calcification assay we determined that extracts from Snus tobacco hindered calcification of osteoblasts derived from pluripotent stem cells early on in their differentiation. Nicotine, a major component of tobacco products, had no measurable effect in the tested concentration range. However, through the extraction of video data, we determined that the tobacco-specific nitrosamine N′-nitrosonornicotine caused a reduction in calcification with similar kinetics as the complete Snus extract. From measurements of actual nitrosamine concentrations in Snus tobacco extract we furthermore conclude that N′-nitrosonornicotine has the potential to be a major trigger of developmental osteotoxicity caused by Snus tobacco.Snus adversely affects human embryonic stem cell calcification yield.A video-based calcification analysis revealed delayed calcification kinetics.Nicotine is not a major contributor to the osteotoxic effect of Snus.NNN inhibited osteoblast differentiation at concentrations present in Snus.NNN caused delayed calcification similar to that found with Snus.