Stage separation is a critical technical issue for developing two-stage-to-orbit (TSTO) launch systems with widebody carrier aircraft that use air-breathing propulsion and launch vehicle stages that use rocket propulsion. During conceptual design phases, this issue can be addressed with a combination of engineering methods, computational fluid dynamics simulations, and trajectory analysis of the mated system and the launch vehicle after staging. The outcome of such analyses helps to establish the credibility of the proposed TSTO system and formulate a ground-based test programme for the preliminary design phase. This approach is demonstrated with an assessment of stage separation from the shuttle carrier aircraft. Flight conditions are determined for safe mated flight, safe stage separation, and for the launch vehicle as it commences ascending flight. Accurate assessment of aerodynamic forces and moments is critical during staging to account for interference effects from the proximities of the two large vehicles. Interference aerodynamics have a modest impact on the separation conditions and separated flight trajectories, but have a significant impact on the interaction forces.