You must reply to that boy who wrote you a letter 谁是主语

you
是主语。

You must reply to that boy who wrote you a letter

who wrote you a letter

分句里who 作主语，不可以换。

You must reply to that boy who/whom you wrote a letter.

这句可以，分句中作宾语

不能，定语从句中差主语，而whom只作宾语，who既可作主语也可作宾语，但介词提前时只用whom，如for whom,with whom

这个句子主干是You must reply to sb.然后宾语用的是一个定语从句，that boy是从句的主语，wrote you a letter是定语，修饰that boy.其实就是2个句子：
1.You must reply to that boy.
2.The boy wrote you a letter.
that boy是先行词，在从句里面作主语，所以用表示人的主格who

you 是主语

可以换