A micropropagation protocol was developed for the HS314 rootstock, a hybrid between almond and peach that could be used as an alternative rootstock instead of GF677. Surface-sterilized nodal segments were cultured in a modified DKW medium containing 3% sucrose, 100 mg·L−1 of Phloroglucinol, 0.7% plant agar, and 0.5 mg·L−1 benzyl amino purine (BAP). Explants were transferred to the same culture media supplemented with 0.5, 1, or 2 mg·L−1 BAP and 0, 0.1, or 0.5 mg·L−1 indole butyric acid (IBA) for further shoot proliferation. The maximum number of shoots produced on a medium containing 2 mg·L−1 BAP. Microshoots were transferred to the DKW medium supplemented with 0.5, 1, 2, or 4 mg·L−1 IBA or naphthaleneacetic acid (NAA) for root induction. The highest root number and the greatest root length were gained on a medium containing 2 mg·L−1 IBA. Rooting percentage was improved from 66% to more than 85% by reducing the concentration of DKW salts to half strength. Finally, rooted plantlets were successfully acclimatized and transferred in vivo conditions.