21 Then Jesus said to them again, "I am going away, and you will seek Me, and will die in your sin. Where I go you cannot come."
22 So the Jews said, "Will He kill Himself, because He says, ‘Where I go you cannot come’?"
23 And He said to them, "You are from beneath; I am from above. You are of this world; I am not of this world.
24 Therefore I said to you that you will die in your sins; for if you do not believe that I am He, you will die in your sins."
This probably took place later on the same day as the conversation in the previous verses.
I am going away (v. 21) — according to the purpose and plan of God, to His death
you will seek Me (v.21) — They would look for the Messiah, but having rejected Christ, they wouldn't find Him. Therefore, they would die in their sins.
The Jews didn't understand (v. 22). They thought He spoke of suicide (although in Greek, the sense is that they didn't really expect Him to). He answeres by explaining why they don't understand. But the natural man does not receive the things of the Spirit of God, for they are foolishness to him; nor can he know them, because they are spiritually discerned (1 Corinthians 2:14).
beneath (v. 23) — this world. When we believe, we are born from above.
Verse 24 denies any theory that there is salvation after death for any who didn't believe when alive.
When your terror comes like a storm, and your destruction comes like a whirlwind, when distress and anguish come upon you. Then they will call on me, but I will not answer; they will seek me diligently, but they will not find me. Because they hated knowledge and did not choose the fear of the LORD (Proverbs 1:27-29).
Then He takes up the previous thought of "dying in your sins." What does it mean? Did He mean their individual sins only? It means that the great sin on account of which they die in their sins is unbelief. "For if ye believe not that I am He, ye shall die in your sins." This is still the Truth. No matter how great a sinner a man or woman may be, there is salvation at the door of each by believing on the Lord Jesus Christ, believing that He is the Son of God and died for the ungodly. No matter how morally a man and a woman may live, how much good they do, if they reject the Lord Jesus Christ, if they refuse to believe on Him, it is tru of all, as it was true of those Jews in the days of our Lord, "If ye believe not that I am He (the Lord) ye shall die in your sins." — The Gospel of John, by Arno Clemens Gaebelein, page 160