KATANIN is a heterodimeric microtubule-severing AAA ATPase protein, consisting of a catalytic p60 and a regulatory p80 unit, playing significant roles in various cellular processes, such as cell division, cell elongation, and morphogenesis. The mutant fragile fiber 2 (fra2) bares a deletion of the A2329 residue, which leads to a frameshift in the ORF and thus to a premature stop codon. The aberrant protein, which lacks 78 amino acids from the C-terminal region, affects the abovementioned processes in all organs of the plant and therefore fra2 demonstrates a dwarf phenotype. It is known that cortical microtubules regulate cellulose microfibril deposition, and previous studies have also shown that root xylem differentiation in fra2 is compromised. In this study, we examined xylem element differentiation in fra2 and wild-type (Col-0) cotyledons, using the VISUAL culture system in which xylem ontogenesis is induced in vitro. Transverse and longitudinal sections of chemically-fixed and resin-embedded cotyledons of both fra2 and Col-0, before and after induction, were stained with toluidine blue and observed under an optical microscope. Moreover, whole cotyledons were examined after chloral hydrate treatment. The results highlighted that the xylem-differentiated fra2 cambial cells underwent an increased and irregular number of cell divisions. Also, as opposed to wild-type plants, fra2 cotyledons exhibited an undifferentiated central nerve and incomplete vascular branching in their cotyledons, even after the induction of xylogenesis. Furthermore, while the induction of Col-0 plants often led to an increased number of ectopic xylem elements, in fra2 mutants their appearance was sporadic. The above data underline the important role of KATANIN during xylem ontogenesis and differentiation in Arabidopsis thaliana.