Bcl-2 inhibitor venetoclax (VEN)-based regimen is a promising therapeutic option in AML, especially for older and unfit patients. Although a high CR rate can be achieved rapidly, the duration of response is limited, and most patients relapsed in a few months. Considerable effort has been made to investigate the mechanism of resistance to VEN in AML, but the data generated were mainly based on findings in in vitro cell lines. To investigate the microenvironmental contribution to VEN resistance, we performed experiments in vitro co-culturing primary patient samples with mesenchymal stromal cells (MSCs) and in vivo using patient-derived xenograft models (PDX). We found that, without MSC co-culture, VEN in general, effectively induced apoptosis in both relapsed/refractory and treatment-naive samples. However, such effect was significantly blunted in relapsed/refractory samples when co-cultured with MSCs, suggesting a role of microenvironment in mediating resistance to VEN. We treated a PDX model established from a patient who clinically relapsed after VEN/HMA treatment, with the Mcl-1 inhibitor AZD5991 alone and in combination with VEN. Although the combination significantly prolonged survival (Carter B et al, ASH2018), the mice eventually died of the disease. By combining RNA-sequencing and targeted gene sequencing in samples from moribund PDX mice, we found neither upregulation of reported targets that may mediate resistance in cell line screenings nor mutation of BCL2. Instead, we observed that VEN treatment significantly induced a proinflammatory cytokine profile, which includes colony-stimulating factor 1 (CSF1), CSF2, IL1, and IL4. This aberrant expression was even more pronounced in samples from AML that relapsed from the VEN/AZD5991 combination group. Since primary AML cells and macrophages/monocytes in niche possess receptors for these cytokines, our results suggest that AML cells survive apoptogenic stress by a crosstalk involving specific cytokines. In addition, expansion of monocytic subgroups in AML samples was reported to establish VEN resistance. We found that AZD5991 significantly decreased monocytic cell markers, suggesting that Mcl-1 inhibition may effectively eliminate monocytic cells in VEN- resistant/relapsed AML. We, here, identify a novel mechanism underlying VEN/Mcl-1 inhibitor resistance and are investigating whether targeting aberrantly expressed cytokines such as CSF1 may overcome it.