Nanocluster aerosols (NCAs, <3 nm particles) are associated with climate feedbacks and potentially with human health. Our recent study revealed NCA formation owing to the reaction of ozone with human surfaces. However, the underlying mechanisms driving NCA emissions remain unexplored. Squalene is the most abundant compound in human skin lipids that reacts with ozone, followed by unsaturated fatty acids. This study aims to examine the contribution of the squalene-ozone reaction to NCA formation and the influence of ozone and ammonia (NH3) levels. In a climate-controlled chamber, we painted squalene and 6-hexadecenoic acid (C16:1n6) on glass plates to facilitate their reactions with ozone. The squalene-ozone reaction was further investigated at different ozone levels (15 and 90 ppb) and NH3 levels (0 and 375 ppb). The results demonstrate that the ozonolysis of human skin lipid compounds contributes to NCA formation. With a typical squalene-C16:1n6 ratio found in human skin lipids (4:1), squalene generated 40 times more NCAs than did C16:1n6 and, thus, dominated NCA formation. More NCAs were generated with increased ozone levels, whereas increased NH3 levels were associated with the stronger generation of larger NCAs but fewer of the smallest ones. This study experimentally confirms that NCAs are primarily formed from squalene-ozone reactions in ozone-human chemistry.