。
She spoke in a gentle tone.
Literal
She [topic-は] gentle tone [in-で] spoke.
口調 ('tone of voice, manner of speaking') refers specifically to the prosodic feel of speech — its softness, sharpness, briskness — rather than the literal voice or its content. 穏やかな is one of those classically positive na-adjectives describing demeanor: peaceful, calm, mild. The で here marks the manner of the action: 'spoke with [a gentle tone].' Particle で is a workhorse — it can mean 'at,' 'with,' 'by means of,' 'in,' depending on context.