S100A1 and S100B are induced by the SOX trio transcription factors (SOX9, SOX5, and SOX6) in chondrocytes, and inhibit their hypertrophic differentiation in culture. However, functional roles of S100A1 and S100B during in vivo skeletal development are yet to be determined. Here we show that mice deficient of both the S100a1 and S100b genes displayed normal skeletal growth from embryonic stage to adulthood. Although no compensatory upregulation of other S100 family members was observed in S100a1/S100b double mutants, the related S100a2, S100a4, S100a10, and S100a11 were expressed at similarly high levels to S100a1 and S100b in mouse primary chondrocytes. Furthermore, overexpression of these other S100 members suppressed the hypertrophic differentiation of chondrocytes in vitro as efficiently as S100A1 and S100B. Taken together, the present study demonstrates that S100A1 and S100B are dispensable for endochondral ossification during skeletal development, most likely because their deficiency may be masked by other S100 proteins which have similar functions to those of S100A1 and S100B.