Abstract Extracellular vesicles (EVs) are pivotal in intercellular communication, impacting diverse physiological and pathological processes. Current in vitro EV biogenesis studies often utilize pharmacological inhibitors, inducing off‐target effects and overlooking cell‐specific production nuances. Addressing these limitations, we utilized CRISPR/Cas9 to generate heterozygous full‐body and conditional sphingomyelin phosphodiesterase 3 (Smpd3) knockout (KO) transgenic mice. Smpd3 , also known as neutral sphingomyelinase 2 (nSMase2), triggers membrane curvature through sphingomyelin hydrolysis to ceramide, thereby influencing exosome release. Intriguingly, Smpd3 deficiency demonstrated no impact on EV release both in vitro and in vivo, underscoring its potential cell‐type‐specific role in EV biogenesis. Notably, bone marrow derived macrophages (BMDMs) did exhibit reduced EV release upon Alix deletion. Our findings open avenues for subsequent inquiries, enriching our knowledge of EV biogenesis and illuminating intercellular communication in health and disease.