Membrane fusion is a fundamental biophysical process essential for cellular activities, including neurotransmitter release, viral entry, and intracellular trafficking [1, 2]. Despite its importance, the energetic pathways and the influence of lipid composition on fusion remain elusive [3, 4]. In this study, we performed coarse-grained molecular dynamics (CG MD) simulations to quantify the free energy landscape of membrane fusion, specifically examining the regulatory role of cholesterol. To quantitatively characterize the fusion process, we employed CG MD simulations based on the SPICA force field [5]. Free energy profiles were calculated using umbrella sampling along a reaction coordinate describing the intermembrane states toward stalk formation. The resulting potential of mean force was reconstructed using the weighted histogram analysis method, enabling direct comparison of fusion energetics across different lipid compositions and cholesterol concentrations. Our simulations demonstrate that the presence of cholesterol modulates the transition states of the fusion process. Specifically, we found that cholesterol increases the free energy barrier required to form a stalk, which connects two separate membranes. Further analyses suggest that the membrane flexibility was a key factor that regulates the membrane fusion. Furthermore, we investigated the spatial preferences of fusion in complex lipid environments. In a ternary lipid mixture exhibiting phase separation, our results reveal that the fusion stalk is preferentially formed in the liquid-disordered domain. This localization highlights the critical interplay between membrane fluidity, domain organization, and the mechanical work required for fusion. Together, our findings provide a mechanical rationale for why cellular membranes maintain specific lipid microdomains to facilitate or inhibit membrane remodeling events.