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Hi there ๐ it looks like you're trying to create a Transfer, which does require that the account you're transferring from has enough balance to cover the amount of the Transfer.
If this was a live payment, then yes you will need to wait for those funds to become available before you can Transfer them. In addition to waiting for funds to settle, automatic payouts can also send your entire Stripe balance to your associated bank account resulting in your Stripe balance not having enough funds to make a Transfer.
how long do they take to become available
That is something we're less familiar with in this forum (we primarily help developers with their questions about how to use the Stripe API) so you may want to consult our support team for a more definitive answer:
https://support.stripe.com/?contact=true
My understanding though is that it depends on the type of payment method that was used, and what country your account is based in. I believe the country is the primary driver:
https://stripe.com/docs/payouts#standard-payout-timing
With the type of payment method being a secondary driver if you're accepting payment methods that take longer to settle, such as ACH:
https://stripe.com/docs/payments/ach-debit#timing
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