Heterotopic ossification (HO), acquired or hereditary, is the formation of true bone outside the normal skeleton. Although the lineages of cells contributing to bone formation during normal development are well defined, the precise lineages of cells that contribute to HO are not clear. This study utilized Cre-lox based genetic lineage tracing to examine the contribution to HO of cells that expressed either FoxD1 or Glast. Both lineages contributed broadly to different normal tissues, and FoxD1-cre labeled cells contributed to normal bone formation. Despite the similarity in labeling patterns of normal tissues, and the significant contribution of FoxD1-cre labeled cells to normal bone, only Glast-creERT labeled progenitors contributed significantly to HO at all stages, suggesting that the cell populations that normally contribute to physiological bone formation, such as the Foxd1-cre labeled cells, may not participate in pathological HO. Further, identification of Glast-expressing cells as precursors that give rise to HO should help with the molecular targeting of this population both for the prevention and for the treatment of HO.