Personally, I use type aliases in cases where I need to describe very dynamic types that make intuitive sense but are too wordy to pattern-match visually. So you might decompose that example like:

    FileSourceType = Union[basestring, file]
    FileSpecType = Union[
        # (filename, file_source)
        Tuple[basestring, Optional[FileSourceType]],
        # (filename, file_source, content_type)
        Tuple[basestring, Optional[FileSourceType], Optional[basestring]],
        # (filename, file_source, content_type, custom_headers)
        Tuple[basestring, Optional[FileSourceType], Optional[basestring], Optional[Headers]]
    ]

    ...

    files: Optional[
        Union[
            Mapping[basestring, FileSpecType], 
            Iterable[Tuple[basestring, FileSpecType]
        ]
    ]

You theoretically lose some "glance value" because now you have to look in two places for the type...but in practice, I think you can figure it out a lot easier than the original example. Obviously you don't want to do this in simple cases, but it can make pathological cases like the above a lot easier to chew on.