Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP_i). Deletion of the TNAP gene (Akp2) in mice results in hypophosphatasia characterized by elevated levels of PP_i and poorly mineralized bones, which are rescued by deletion of nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) that generates PP_i. Mice deficient in NPP1 (Enpp1^−/−), or defective in the PP_i channeling function of ANK (ank/ank), have decreased levels of extracellular PP_i and are hypermineralized. Given the similarity in function between ANK and NPP1 we crossbred Akp2^−/− mice to ank/ank mice and found a partial normalization of the mineralization phenotypes and PP_i levels. Examination of Enpp1^−/− and ank/ank mice revealed that Enpp1^−/− mice have a more severe hypermineralized phenotype than ank/ank mice and that NPP1 but not ANK localizes to matrix vesicles, suggesting that failure of ANK deficiency to correct hypomineralization in Akp2^−/− mice reflects the lack of ANK activity in the matrix vesicle compartment. We also found that the mineralization inhibitor osteopontin (OPN) was increased in Akp2^−/−, and decreased in ank/ank mice. PP_i and OPN levels were normalized in [Akp2^−/−; Enpp1^−/−] and [Akp2^−/−; ank/ank] mice, at both the mRNA level and in serum. Wild-type osteoblasts treated with PP_i showed an increase in OPN, and a decrease in Enpp1 and Ank expression. Thus TNAP, NPP1, and ANK coordinately regulate PP_i and OPN levels. The hypomineralization observed in Akp2^−/− mice arises from the combined inhibitory effects of PP_i and OPN. In contrast, NPP1 or ANK deficiencies cause a decrease in the PP_i and OPN pools that leads to hypermineralization.