The maintenance of telomere length is essential for the indefinite proliferation of cancer cells. This is most often achieved by the activation of telomerase; however, a substantial number of cancers lack detectable telomerase activity and are classified as using an alternative lengthening of telomeres (ALT) pathway. We showed recently that ALT cells have a high level of extrachromosomal telomeric circles (t circles) that may be a specific marker of the ALT phenotype. The mechanism underlying t circle production and the requirement of t circles in ALT remain unclear. Understanding the specific requirements of ALT is key to developing diagnostic tools and therapies that target this pathway and is critical for the treatment of cancers in which ALT is prevalent, including cancers of neuroepithelial and mesenchymal origin. In this study, we used short hairpin RNAs directed at either Xrcc3 or Nbs1, two proteins involved in the homologous recombination pathway, to determine the role of these proteins in t circle production and the requirement of t circles in maintaining the ALT pathway. We show that Xrcc3 and Nbs1 are indeed required for the production of t circles in human ALT. However, these cells continue to proliferate in the absence of t circles, suggesting that they are not required for the survival of ALT cells.