Quantitative label-free proteome analysis of iPSC-derived immortalized megakaryocytes (imMKCLs), sorted by cell size and culture duration into six fractions, was performed in this project. Following tryptic digestion, peptide samples were analyzed by liquid chromatography–mass spectrometry (LC-MS). For each run, 3 µg of peptide mixture was injected into an Ultimate 3000 nanoUHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a trap column (Acclaim PepMap100, PN:164564, Thermo Fisher Scientific) in buffer A (0.1% formic acid) and separated on an analytical column (nanoESI column, 15 cm × 75 µm, NTCC-360/75-3-155, Nikkyo Technos, Tokyo, Japan) maintained at 35°C. Separation was performed at 300 nL/min using a linear gradient from 4% to 40% buffer B (100% acetonitrile, 0.1% formic acid) over 100 min, followed by a 1 min increase to 90% buffer B and a 5 min wash at 90% buffer B. Mass spectrometry was performed using data-dependent acquisition. Detailed MS acquisition parameters are summarized in Table S10. Raw data were processed using Proteome Discoverer Software 2.5 (Thermo Fisher Scientific) against the UniProt human proteome database (UP000005640; http://UniProt.org). Peptide-spectrum matches were validated using Percolator, and protein identifications were filtered at a false discovery rate of <1%. Label-free quantification was performed based on precursor ion intensities.