Scottish schools must provide separate toilets for boys and girls, the government has said.
New guidance issued by Holyrood ministers says that facilities must be made available on the basis of biological sex, but that schools can also provide gender neutral toilets.
Schools had previously been told that transgender pupils could use whatever toilets they felt most comfortable in.
Education Secretary Jenny Gilruth said the guidance provided "clarity" following two landmark court rulings.
In April, the Supreme Court ruled that a woman is defined by biological sex under equalities law.
Two months later, a judge ordered that six Scottish schools must provide single-sex toilets for pupils.
The updated Scottish government guidance says that under the law toilet facilities must be "made available on the basis of biological sex".
It also says educational authorities should consider toilet arrangements for transgender pupils, such as "gender neutral provision".
In its previous guidance, published in 2021, the government said there was no law that stated "only people assigned male at birth can use men's toilets and changing rooms, or that only people assigned female can use women's toilets and changing rooms".
It also said that in PE classes organised by sex, transgender pupils should be able to take part with the group that matches their "gender identity".
However, this line is removed in the new document.
The new guidance says that it is important to support and listen to young people who say that they "want to live as a boy although their biological sex was female, or they now want to live as a girl, although their biological sex was male".
Denying this could have "a detrimental impact on the young person's wellbeing, relationships and behaviour and this is often clearly apparent to teachers, parents and carers", the guidance says.
The government says that "being alert to gender stereotypes and being aware that not every child will identify as their biological sex will help create an environment in which all young people can flourish".
It suggests that primary schools should ensure that play and learning is "gender neutral" and participate in LGBT History Month and Transgender Day of Visibility.
It also suggests using books that challenge gender stereotypes, and ensuring feedback is neutral, so that "boys and girls aren't praised for different things".
The guidance warns that "forcing transgender young people to wear clothes which do not match their gender identity can be distressing for them" and could be a breach of the Equality Act.
It says that educational staff "should take care not to 'out' a young person by using a pronoun which differs from the one which the young person usually uses in public".
The government adds: "Similarly, staff and young people should avoid misgendering a transgender young person."