T-DM1 inhibits megakaryocyte differentiation and proplatelet production by disrupting microtubule organization. The rate/extent of megakaryocyte differentiation and proplatelet production was examined in at least 3 different primary cultures after incubation with 100 μg/mL of T-DM1, 5B6-DM1 (trastuzumab specificity control), and trastuzumab Ab control. Quantitative analysis of fetal liver cell and round megakaryocyte cultures was performed on the IncuCyte system using investigator-coded software. (A) Quantification of the rate and extent of megakaryocyte differentiation from mouse fetal liver cell culture over 96 hours. Inserts demonstrate representative images taken on day 4 of culture. (B) Quantification of the rate and extent of proplatelet production by mature megakaryocytes over 48 hours. Inserts demonstrate representative images taken after 10 hours of culture. (C) Megakaryocytes incubated with T-DM1 showed fewer individual proplatelet extensions per megakaryocyte and reduced proplatelet branching relative to trastuzumab control. Quantification of proplatelet extensions per megakaryocyte was performed at 6 hours for trastuzumab culture and at 10 hours for T-DM1 culture to account for the delay in proplatelet production. Quantification of proplatelet length before branching was performed at 24 hours for both cultures. (D) Mouse culture megakaryocytes were retrovirally transfected to express EGFP–β1-tubulin. Inserts demonstrate representative images taken before and after proplatelet production. Megakaryocytes incubated with T-DM1 showed thicker microtubules relative to trastuzumab control as demonstrated by quantification of mean fluorescence intensity across the shaft (t = 24 hours). Statistical significance was established using a 1-tailed Student t test for paired samples. *P < .05; **P < .01. Error bars represent 1 SD about the mean for at least 28 independent samples.