The formation of nano-scale clusters (nanoclusters) prior to the precipitation of the strengthening β" phase significantly influences the two-step aging behavior of Al-Mg-Si alloys. In this work, two types of nanoclusters are found to be formed at different temperatures. The characterization of these two nanoclusters has been performed from the viewpoints of composition and thermal stability using a three-dimensional atom probe (3DAP) and differential scanning calorimetry (DSC). Mg-Si co-clusters formed at room temperature (RT), Cluster(1), play a deleterious role in the subsequent formation of the β" phase because of the high thermal stability even at the bake-hardening (BH) temperature of 443K. In contrast, the nanoclusters formed by pre-aging at 373K, Cluster(2), are effective in the formation of the refined β", suggesting that Cluster(2) transforms more easily into the β" phase than Cluster(1). The quantitative estimation of the chemical compositions of the two nanoclusters suggests that the Mg/Si ratio is one of the key factors in addition to the internal structures consisting of Si, Mg and probably vacancies. The detailed two-step aging mechanism in Al-Mg-Si alloys is proposed based on the characteristics of the two types of nanoclusters.