Background: Dedifferentiated liposarcomas (DDLPS) are malignant adipocytic cancers characterized by an amplification of the cell cycle regulatory gene, CDK4. Palbociclib, a CDK4/6-inhibitor, is a standard of care treatment; however, innate and acquired resistance to the drug limits efficacy in patients with DDLPS. Molecular mechanisms and biomarkers of palbociclib resistance have been posited in non-mesenchymal tumors but are poorly understood in DDLPS. To this end, we developed palbociclib-resistant DDLPS cell lines derived from several DDLPS patient samples to better characterize these phenotypic and genomic differences. Methods: Three human CDK4 amplified DDLPS cell lines (224A, 246, 863) and one control liposarcoma control cell line without CDK4 amplification and RB loss (LiSa2) were brought into culture. Each cell line was intermittently treated with palbociclib at their respective IC₂₅for 6 months to develop resistance in long-term treated cell lines. Surviving cells were allowed to recover for at least 2 weeks before retreatment. Parental lines were grown as controls in tandem with the developing resistant lines to compare molecular changes in response to palbociclib. Cell viability was measured by XTT, cell cycle analysis was measured with Flow cytometry using a PI stain, and protein expression was measured using whole cell lysates for Western blotting. Results: The four cell lines had IC50s to palbociclib ranging from 15.0, 14.8, 12.0, and 11.4 µM for LiSa-2, 224A, 246, and 863 cell lines respectively. Long-term treated strains demonstrated consistent reductions in the measured IC50s (8.99, 7.32, 8.27, and 7.08 µM respectively). After treatment with 10 µM of palbociclib for 48 hours, two parental cell lines showed a greater increase in cells in the G1 fraction than long-term treated cells: LiSa-2 (-2.4% increase in parental vs -16.5% increase in long-term treated) and 863 (16.2% vs 11.7%). 224A and 246 demonstrated the opposite where parental cell lines had fewer cells in G1 after treatment compared to long-term treated cell: 224A (12.6% vs. 20.5%) and 246 (12.6% vs. 21.6%). Western blots indicated that the initial response to palbociclib in parental cell lines decreased pRb protein levels and increased Cyclin E and CDK6. After 6 months of treatment for all cell lines, a significant increase in pRb and Cyclin E were observed in the long-term treated cell lines and no changes were seen in CDK4, Cyclin D, nor p16. Conclusions: Here we present the development of two novel, palbociclib resistant, DDLPS cell lines and two long-term treated cell lines. Our resistant cell lines indicated that the ability to bypass the G1/S-checkpoint in the presence of CDK4/6-inhibitors may result from increased phosphorylation of Rb and increased total of Cyclin E. Next generation sequencing of RNA expression in parental and resistant lines are underway.