Learn Spanish (Latin America): ¿Qué es eso en tu mochila?

eso is the neuter (genderless) form of "that". We use it when we are pointing at something but do not know what it is yet. Since we do not know the noun for the object, we cannot know if it is masculine or feminine. For unknown objects, we always use the neuter eso.

Doesn't the "o" at the end of eso mean it is a masculine word?

It is a very common trap! For demonstrative words like this and that, the masculine forms are actually ese (that) and este (this). The forms ending in "o" (eso, esto) are specifically the neuter forms used for unknown things or general ideas.

Since the thing is located in the backpack, why do we use es instead of está?

The core of this sentence is asking to identify an object ("What is that thing?"), which always requires the verb ser (es). Even though the sentence adds a location (en tu mochila), the main goal is identifying what the thing is, not asking where it is.

Why does tu not have an accent mark here?

In Spanish, tu without an accent mark is the possessive word meaning "your", as used here in tu mochila (your backpack). If it had an accent mark (tú), it would be the subject pronoun meaning "you".