Organophosphate esters (OPEs) are used widely as flame retardants and plasticizers but much remains unknown about their potential toxicity. Previously, we reported that 4 individual OPEs suppress endochondral ossification in murine limb bud cultures. However, real-life exposure is to complex OPE mixtures. In the present study, we tested the hypothesis that a Canadian household dust-based OPE mixture will affect endochondral ossification in gestation day 13 CD1 mouse embryo limb buds expressing fluorescent markers for the major cell populations involved in the process: collagen type II alpha 1-enhanced cyan fluorescent protein (proliferative chondrocytes), collagen type X alpha 1-mCherry (hypertrophic chondrocytes), and collagen type I alpha 1-yellow fluorescent protein (osteoblasts). Limbs were cultured for 6 days in the presence of vehicle or dilutions of the OPE mixture (1/1 000 000, 1/600 000, and 1/300 000). All 3 OPE mixture dilutions affected cartilage template development and the progression of endochondral ossification, as indicated by the fluorescent markers. The expression of Sox9, the master regulator of chondrogenesis, was unchanged, but the expression of Runx2 and Sp7, which drive chondrocyte hypertrophy and osteoblastogenesis, was dilution-dependently suppressed. RNA-seq revealed that exposure to the 1/300 000 dilution of the OPE mixture for 24 h downregulated 153 transcripts and upregulated 48 others by at least 1.5-fold. Downregulated transcripts were enriched for those related to the immune system and bone formation. In contrast, upregulated transcripts were enriched for those with stress response functions known to be regulated by ATF4 activation. Thus, exposure to the mixture of OPEs commonly found in house dust may have adverse effects on bone formation.