Yes. The Simulator allows you to use two Single-Player USB Interfaces in Two-Player mode. Note: You must make sure to use the Select Interface button to change the Interface type from a Single-Player USB Interface to the Second-Player USB Interface as interface FS One defaults to the Single-Player interface type.
In the above, “Single-Player USB Interface” refers to the original FS One USB Interface supplied with the simulator package. “Second-Player USB Interface” refers to the red second-player FS One USB Interface available as an add-on purchase when it was available. It is shown below.
Realize that the second player can use any USB interface that can be recognized by Windows, e.g. a 3rd party controller/interface such as the MileHighWings USB Interface attached to a transmitter.