Leaves excised from in vitro obtained plantlets of Aeonium haworthii Webb & Berthel. were used as explants to establish a protocol for adventitious shoots formation, rooting, plantlet strengthening, and acclimatization. For culture establishment, leaf explants disinfected with 0.1% HgCl₂ for 8 min showed the best overall response, with the highest level of aseptic explants (72.3%). Adventitious shoots formation was markedly affected by basal medium strength and plant growth regulators combinations and concentrations. Under a fixed plant growth regulator background, one-third-strength MS medium promoted higher adventitious shoot formation than full-strength MS, half-strength MS, or one-quarter-strength MS medium. In the single-factor experiments, BA and NAA were more favorable for adventitious shoots formation than KT and 2,4-D, whereas 2,4-D mainly induced callus formation. In the full-factorial experiment, one-third-strength MS medium supplemented with 0.5 mg l^-1 BA and 0.5 mg l^-1 NAA promoted compact rosette-like shoot formation and showed the best regeneration performance. Shoots with uniform growth were then transferred to one-third-strength MS medium supplemented with 0.5 mg l^-1 NAA for rooting and plantlet strengthening. Following acclimatization in a humus soil and perlite mixture (3 : 1, v : v), the plantlets showed 100% survival after 60 days.