“gain by”

My first impulse is to get rid of the "from," if only because it might raise the expectation of a "to" later! (As in "from soup to nuts.") I think "by" is the best alternative. We learn by doing, so why not gain expertise by negotiating, meeting, and presenting? :-)

I don't think you need the comma after Mexico, though.

You can use by altough 'via' would be a more appropriate word to use.

I think you included the seeds of your own answer. as you suggested,
"...you could really gain "by" something" does sound awkward.
but:
"...you could really gain "from" something"
sounds ok.
Well, consider this:
"...you could really gain "by" DOING something"
sounds ok, doesn't it?
I'm no English professor, but I think this is the paradigm:
If the cause of your gain is a noun, then you gain "from" it, but if the cause of your gain is a verb, then you gain "by" it.
example:
I gained a lot from excercise.
I gained a lot by excercising.

I think the cause of the confusion could be eliminated by moving the phrase "as a project director in Ecuador and in Mexico," to the beginning of the sentence (deleting the "in" before "Mexico," where it is unnecessary), and changing the "from" to "by," so that it would read as follows:

"As a project director in Ecuador and Mexico, I gained expertise in effective communication by negotiating in professional settings, meeting with my staff, and presenting to volunteers."