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Formation of intrinsic acceptor defects in ZnO is rare due to the low formation energy of donors. Understanding this phenomenon is of interest for the fabrication of high quality light emitting diodes. Herein, we examine the temperature dependent formation of defects in nanocrystalline ZnO through a combination of X-ray excited optical luminescence (XEOL) together with X-ray absorption near edge structures (XANES) and electron spin resonance (ESR). Certain defects are shown to form under low temperature and are unstable above 700 °C. These defects have high g-values characteristic of acceptors and short spin-lattice relaxation times. XEOL measurements show that acceptor defects with a characteristic red luminescence are also formed under these conditions. Low g-value (donor) defects forming at temperatures >700 °C are shown to have spin-lattice relaxation characteristic of nonradiative recombination centers.