Tumor development relies on metabolic reprogramming, offering therapeutic opportunities. Glutamine (Gln) is an essential amino acid crucial for providing the necessary elements and serving as a readily available source of carbon and nitrogen. It plays a pivotal role in supporting biosynthesis, energetics, and maintaining cellular homeostasis, particularly in Gln-dependent cancers like acute myeloid leukemia (AML). Crisantaspase, Erwinia-derived asparaginase, disrupts Gln supply and has shown promise in preclinical and clinical models of AML. In our AML cell experiments with crisantaspase, we observed increased serine (Ser) levels, suggesting AML cells activate de novo Ser biosynthesis in response to crisantaspase. We hypothesized that inhibiting this pathway would enhance AML cell death in synergy with crisantaspase. Short-acting crisantaspase (Rylaze) inhibited the proliferation of multiple AML cell lines with pharmacologic relevant IC50 values (0.001 to 0.08 µg/mL). Reducing media Gln concentration in cell culture media led to dose-dependent decreases in AML cell proliferation, confirming AML's sensitivity to Gln depletion. Intriguingly, Gln withdrawal and crisantaspase treatment upregulated Ser levels in the media and PHGDH expression. We utilized CRISPR-Cas9 to knock out phosphoglycerate dehydrogenase (PHGDH), a key Ser biosynthesis enzyme, in AML cell lines. We then assessed how PHGDH knockout cells responded to Gln depletion, either through Gln withdrawal or crisantaspase treatment. AML cells lacking PHGDH exhibited significantly increased sensitivity to crisantaspase, with IC50 values ranging from 1.34 to 6.59 picogram/mL, approximately 250-fold lower than parental AML cells. Furthermore, combining BI4916, a PHGDH inhibitor, with Rylaze in AML cell lines and patient samples produced synergistic effects. This combination treatment increased lactate dehydrogenase (LDH) release, decreased glutathione (GSH) production and altered protein synthesis, as evidenced by changes in eIF4E/4E-BP1 mRNA translation-initiation factor proteins and decreased clonogenic ability of AML cells. High-throughput transcriptomic analysis unveiled distinct gene expression differences between the control and combination treatment groups in AML, suggesting the involvement of various potential pathways such as cell death, energy production and immune-related pathways. These findings imply significant translational potential, especially by leveraging clinically available crisantaspases and other Gln modulators in clinical practice.