Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1^+/− and compound Ext1^+/−/Ext2^+/− mice. Like Ext2^+/− mice described previously (Stickens et al. Development 132:5055), Ext1^+/− mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1^+/−Ext2^+/− exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1^+/−Ext2^+/− chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1^f/+Col2Cre and Ext1^f/+Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.