The automated laser-based hematology analyzer Sysmex XT-2000iV™ provides a 5-part differential count and specific cytograms that are of great interest for large veterinary laboratories. The aim of the study was to validate the Sysmex XT-2000iV compared to the laser-based hematology analyzer ADVIA® 2120 and manual differential in dogs, cats, and horses as well as the impact of anticoagulant (heparin, ethylenediamine tetra-acetic acid [EDTA], and citrate) and storage at 22°C and 4°C. Consecutive fresh K(3)-EDTA blood samples from 216 cats, 314 dogs, and 174 horses were included. The impact of anticoagulant and sample storage was assessed in specimens obtained from an additional 9 cats, 10 dogs, and 10 horses. Agreement between both analyzers was excellent to good except for monocytes and canine reticulocytes. Spearman rank correlation coefficients (r (s)) between Sysmex XT-2000iV and manual differential were good to fair and ranged from 0.91 (cat lymphocytes) to 0.44 (cat monocytes). Hematocrit value (Hct), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC; all: P < 0.001), and mean corpuscular volume (MCV; P < 0.01) were higher in canine citrated blood compared to heparin and EDTA. In cats, lymphocytes and monocytes were lower in heparinized blood compared to EDTA (P < 0.05), whereas in horses no significant effect was seen. Regarding storage time and temperature, white and red blood cell counts, hemoglobin, and MCH were stable. Hct, MCV, and MCHC were influenced by erythrocyte swelling. Differential count remained stable for 24 hr (22°C) and nearly 72 hr (4°C) except for monocytes. The overall performance of the Sysmex XT-2000iV was excellent and compared favorably with that of the ADVIA 2120. A special strength was the excellent detection of feline eosinophils.