Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.