Background: ASXL1 and the cohesin complex (STAG2, RAD21, SMC1A, and SMC3) are commonly mutated chromatin regulators with significant clinical implications in AML. The ASXL1-cohesin interactome regulates gene expression through chromatin accessibility via ASXL1’s cohesin binding motif (CBM). ASXL1 variants are most commonly located in the ASXM1 domain and onwards, and characteristically lead to loss of the PHD domain. Gain-of-functions in truncated ASXL1 are suggested to increase catalytic activity of BAP1, which binds the ASXH domain at AA 351, and to gain an interaction with BRD4, which binds somewhere between the ASXN and ASXH domains, to drive H3K4Me3 and H2AK119Ub. Methods: 2463 suspected AML patient bone marrow, peripheral blood, or FFPE tissue samples were evaluated using an all exon amplicon-based 27 gene NGS panel. Patients with a VAF <10% in ASXL1 were excluded to avoid reporting artifacts, particularly in variant c. 1934dup. Statistics were performed using Fisher’s exact test. Results: Mutations in STAG2-mutated patients were enriched for sAML, as evidenced by the higher number of mutations in ASXL1, SRSF2, and BCOR (associated with sAML) compared to NPM1, DNMT3A, and PTPN11 (pAML). STAG2 mutations were found in 173 samples representing 93.5% of cohesin mutations. Of all ASXL1 mutations (VAF 10.1– 54.5%; median 32.2%) 4.0% occurred in the CBM. While 23.5% of samples with mutations outside ASXL1 CBM had concomitant mutations in STAG2, none of the 18 samples with CBM mutations (VAF 11.3 – 51.7%; median 42.5%) had any cohesin gene mutation (P = 0.0174). The proportion of BCOR (27.8% vs 9.2%; P = 0.024) and CEBPA (27.8% vs 8.2%; p = 0.016) mutated patients in the CBM+ group was significantly higher than the CBM- group. JAK2 (16.7% vs 5.4%), KRAS (22.2% vs 13.6%), EZH2 (22.2% vs 13.6%), and RUNX1 (38.9% vs 27.7%) mutations were also higher though not significantly in this group. Mutations throughout all of ASXL1, the 13 amino acids after the CBM, and hotspot variants all had STAG2 mutations at a frequency of 20.9-44.4%, further suggesting mutual exclusivity. Conclusions: STAG2 mutations and mutations in the CBM were mutually exclusive events and harbored different co-mutation frequencies. In compromised ASXL1 CBM cases, BCOR and CEBPA transcriptional regulators are significantly more mutated, but in cases of ASXL1 mutation outside the CBM, cohesin mutations are preferred, suggesting alternative chromatin accessibility mechanisms driving leukemogenesis. This observation has not been previously reported in the literature to our knowledge.